This study aimed to examine the anti-proliferative effects of α-, γ- and δ-tocotrienols (αT3, γT3 and δT3), and α-tocopherol on 3T3-L1 adipocytes. Results showed that compared with other vitamin E analogues, γT3 demonstrated the most potent anti-proliferative effect on 3T3-L1 cells. It significantly caused a reduction in mitochondrial membrane potential (Δψm) and an increase in ROS formation, as well as inducing cell apoptosis and cell cycle arrest at S phase. Further studies showed that it down-regulated Bcl-2 and PPAR-γ expression, suppressed Akt and ERK activation and phosphorylation, and caused cytochrome c release from mitochondria to cytosol, whereas it up-regulated CD95 (APO-1/CD95) and Bax expression, and caused caspase-3 and JNK activation, PARP cleavage and AMPK phosphorylation. Pretreatments with caspase-3 (z-DEVD-fmk) and AMPK (CC) inhibitors significantly suppressed the γT3-induced ROS production and cell death. Caspase-3 inhibitor also efficiently blocked CD95 (APO-1/CD95) and Bax expression, caspase-3 activation and PARP cleavage, whereas antioxidant N-acetyl-L-cysteine, AMPK inhibitor and AMPK siRNA effectively blocked the AMPK phosphorylation. Taken together, these results conclude that the potent anti-proliferative and anti-adipogenic effects of γT3 on 3T3-L1 adipocytes could be through the Bax-mediated mitochondrial and AMPK signaling pathways.
Publications
Rice varietal differences in bioactive bran components for inhibition of colorectal cancer cell growth.
Forster GM, Raina K, Kumar A, Kumar S, Agarwal R, Chen MH, Bauer JE, McClung AM, Ryan EP.
Food Chem. 2013 Nov 15;141(2):1545-52. Epub 2013 Apr 17.
Summary
Rice bran chemical profiles differ across rice varieties and have not yet been analysed for differential chemopreventive bioactivity. A diverse panel of seven rice bran varieties was analysed for growth inhibition of human colorectal cancer (CRC) cells. Inhibition varied from 0% to 99%, depending on the variety of bran used. Across varieties, total lipid content ranged 5-16%, individual fatty acids had 1.4- to 1.9-fold differences, vitamin E isoforms (α-, γ-, δ-tocotrienols, and tocopherols) showed 1.3- to 15.2-fold differences, and differences in γ-oryzanol and total phenolics ranged between 100-275ng/mg and 57-146ngGAE/mg, respectively. Spearman correlation analysis was used to identify bioactive compounds implicated in CRC cell growth inhibitory activity. Total phenolics and γ-tocotrienol were positively correlated with reduced CRC cell growth (p<0.05). Stoichiometric variation in rice bran components and differential effects on CRC viability merit further evaluation elucidate their role in dietary CRC chemoprevention.
Effect of the tocotrienol-rich fraction on the lifespan and oxidative biomarkers in Caenorhabditis elegans under oxidative stress.
Aan GJ, Zainudin MS, Karim NA, Ngah WZ.
Clinics (Sao Paulo). 2013; 68(5): 599–604.
Summary
OBJECTIVE:
This study was performed to determine the effect of the tocotrienol-rich fraction on the lifespan and oxidative status of C. elegans under oxidative stress.
METHOD:
Lifespan was determined by counting the number of surviving nematodes daily under a dissecting microscope after treatment with hydrogen peroxide and the tocotrienol-rich fraction. The evaluated oxidative markers included lipofuscin, which was measured using a fluorescent microscope, and protein carbonyl and 8-hydroxy-2′-deoxyguanosine, which were measured using commercially available kits.
RESULTS:
Hydrogen peroxide-induced oxidative stress significantly decreased the mean lifespan of C. elegans, which was restored to that of the control by the tocotrienol-rich fraction when administered before or both before and after the hydrogen peroxide. The accumulation of the age marker lipofuscin, which increased with hydrogen peroxide exposure, was decreased with upon treatment with the tocotrienol-rich fraction (p<0.05). The level of 8-hydroxy-2′-deoxyguanosine significantly increased in the hydrogen peroxide-induced group relative to the control. Treatment with the tocotrienol-rich fraction before or after hydrogen peroxide induction also increased the level of 8-hydroxy-2′-deoxyguanosine relative to the control. However, neither hydrogen peroxide nor the tocotrienol-rich fraction treatment affected the protein carbonyl content of the nematodes.
CONCLUSION:
The tocotrienol-rich fraction restored the lifespan of oxidative stress-induced C. elegans and reduced the accumulation of lipofuscin but did not affect protein damage. In addition, DNA oxidation was increased.
γ-Tocotrienol Attenuates Triglyceride through Effect on Lipogenic Gene Expressions in Mouse Hepatocellular Carcinoma Hepa 1-6
Burdeos, GC, Nakagawa K, Watanabe A, Kimura F, Miyazawa T.
J Nutr Sci Vitaminol, 2013; 59(2):148-151,
Summary
Vitamin E is the generic name for tocopherol (Toc) and tocotrienol (T3), which have saturated and unsaturated side chains, respectively. Such differences allow T3 to be different from Toc in terms of their functions. T3 has been known to attenuate cholesterol (Cho) level by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoAR). Recent reports also showed the efficacy of T3 in improving triglyceride (TG) profiles in both in vivo and in vitro studies. However the mechanism involved in this biological activity is still unclear and needs to be further investigated. In the present study, we elucidated the effect of γ-T3 on lipid levels and lipogenic gene expressions in mouse hepatocellular carcinoma Hepa 1-6. γ-T3 showed attenuation of TG through effect on fatty acid synthas, sterol regulatory element-binding transcription factor 1, stearoyl CoA desaturase 1, and carnitine palmitoyl. In contrast, the Cho level remained unchanged. These results expanded our previous finding of lipid-lowering effects of T3, especially for TG. Therefore, T3 is a potential lipid-lowering compound candidate with realistic prospects for its use as a therapy for lipid-related diseases in humans.
α-Tocopherol does not Accelerate Depletion of γ-Tocopherol and Tocotrienol or Excretion of their Metabolites in Rats
Uchida T, Nomura S, Sakuma E, Hanzawa F, Ikeda S.
Lipids (2013) 48:687–695
Summary: From an enzyme kinetic study using rat liver microsomes, α-tocopherol has been suggested to accelerate the other vitamin E catabolism by stimulating vitamin E ω-hydroxylation, the late limiting reaction of the vitamin E catabolic pathway. To test the effect of α-tocopherol on catabolism of the other vitamin E isoforms in vivo, we determined whether α-tocopherol accelerates depletion of γ-tocopherol and tocotrienol and excretion of their metabolites in rats. Male Wistar rats were fed a γ-tocopherol-rich diet for 6 weeks followed by a γ-tocopherol-free diet with or without α-tocopherol for
7 days. Intake of γ-tocopherol-free diets lowered γ-tocopherol concentrations in serum, liver, adrenal gland, small intestine, and heart, but there was no effect of dietary α-tocopherol on γ-tocopherol concentrations. The level of urinary excretion of γ-tocopherol metabolite was not affected by dietary α-tocopherol. Next, the effect of α-tocopherol on tocotrienol depletion was examined using rats fed a tocotrienol-rich diet for 6 weeks. Subsequent intake of a tocotrienol-free diet with or without α-tocopherol for 7 days depleted concentrations of α- and γ-tocotrienol in serum and tissues, which was accompanied by a decrease in the excretion of γ-tocotrienol metabolite.
However, neither the tocotrienol concentration nor c-tocotrienol metabolite excretion was affected by dietary α-tocopherol. These data showed that dietary α-tocopherol did not accelerate the depletion of γ-tocopherol and tocotrienol and their metabolite excretions, suggesting that the positive effect of α-tocopherol on vitamin E ω-hydroxylase is not sufficient to affect the other isoform concentrations in tissues.
Vitamin E: tocopherols and tocotrienols as potential radiation countermeasures.
Singh VK, Beattie LA, Seed TM.
J Radiat Res, 2013 May 8. [Epub ahead of print]
Summary
Despite the potential devastating health consequences of intense total-body irradiation, and the decades of research, there still remains a dearth of safe and effective radiation countermeasures for emergency, radiological/nuclear contingencies that have been fully approved and sanctioned for use by the US FDA. Vitamin E is a well-known antioxidant, effective in scavenging free radicals generated by radiation exposure. Vitamin E analogs, collectively known as tocols, have been subject to active investigation for a long time as radioprotectors in patients undergoing radiotherapy and in the context of possible radiation accidents or terrorism scenarios. Eight major isoforms comprise the tocol group: four tocopherols and four tocotrienols. A number of these agents and their derivatives are being investigated actively as radiation countermeasures using animal models, and several appear promising. Although the tocols are well recognized as potent antioxidants and are generally thought to mediate radioprotection through ‘free radical quenching’, recent studies have suggested several alternative mechanisms: most notably, an ‘indirect effect’ of tocols in eliciting specific species of radioprotective growth factors/cytokines such as granulocyte colony-stimulating factor (G-CSF). The radioprotective efficacy of at least two tocols has been abrogated using a neutralizing antibody of G-CSF. Based on encouraging results of radioprotective efficacy, laboratory testing of γ-tocotrienolhas moved from a small rodent model to a large nonhuman primate model for preclinical evaluation. In this brief review we identify and discuss selected tocols and their derivatives currently under development as radiation countermeasures, and attempt to describe in some detail their in vivo efficacy.
Redox-inactive analogue of tocotrienol as a potential anti-cancer agent
Yano T, Sato A, Sekine M, Virgona N, Ota M.
Anticancer Agents Med Chem 2013;139(3):496-501
Vitamins are prominent among natural or endogenous compounds that are considered to be beneficial for both prevention and therapy of various human ailments. The vitamin E group of compounds composed of tocopherol and tocotrienol isoforms, has been subsequently proven to have health benefits including antioxidant and related protective properties. However, individual isoforms exhibit a wide-range of antioxidant potencies. Tocotrienol (T3) displays powerful anticancer activity that is often not exhibited by tocopherols, by modulating multiple intracellular signaling pathways associated with tumor cell proliferation and survival. The anticancer effect of T3 remains not fully understood but generally is mediated independently of its antioxidant activity. Further we have synthesized a new redox-inactive analogue of T3, 6-O-carboxypropyl-alpha-tocotrienol (T3E) showing considerable promise for stronger anticancer potency than its mother compound. In this mini-review, we particularly focus upon the anticancer action of the above active components of vitamin E and describe current research on the anticancer effects of T3 irrespective of antioxidant activity.
Optimization of tocotrienols as antiproliferative and antimigratory leads
Behery, F. A.,Akl, M. R.,Ananthula, S.,Parajuli, P.,Sylvester, P. W.,El Sayed, K. A.
Eur J Med Chem 2013;59:329-41
The vitamin E family members gamma- and delta-tocotrienols (2 and 3, respectively) are known natural products with documented anticancer activities. Redox-silent structural modifications, such as esterification, etherification and carbamoylation, of 2 and 3 significantly enhanced their anticancer activities. However, hit-to-lead optimization of tocotrienols and their analogs was yet to be reported at the outset of the project described herein. Subjecting the chroman ring of 2 and 3 to the electrophilic substitution reactions, namely, Mannich and Lederer-Manasse procedures, afforded 42 new products. These included the 3,4-dihydro-1,3-oxazines 3-29 and 35-44, Mannich bases 30-31, and the hydroxymethyl analogs 32-34. Of these, the delta-tocotrienol analogs 8, 11, 18, 24, 25, 27, and 40 inhibited the proliferation of the highly metastatic +SA mammary epithelial cancer cell line, with IC(50) values in the nanomolar (nM) range. In NCI’s 60 human tumor cell line panel, 8, 17, 38, and 40 showed antiproliferative activity, with nM GI(50) values. The delta-tocotrienol analogs 10 and 38 inhibited the migration of the highly metastatic human breast cancer cell line MDA-MB-231 with IC(50) values of 1.3 and 1.5 muM, respectively, in the wound-healing assay. A dose of 0.5 mg/day for 14 days of one of the active analogs, 30, significantly slowed the growth of +SA mammary tumors in the syngeneic BALB/c mouse model, compared to the vehicle- and the parent gamma-tocotrienol-treated control groups. Electrophilic substitution reactions promoted tocotrienols to lead level and can enable their future use to control metastatic breast malignancies.
Effect of annatto-tocotrienols supplementation on the development of mammary tumors in HER-2/neu transgenic mice
Pierpaoli, E.,Viola, V.,Barucca, A.,Orlando, F.,Galli, F.,Provinciali, M.
Carcinogenesis 2013;34(6):1352-60
Tocotrienols (T3), the lesser known isomers of vitamin E, have been reported to possess anticancer activity both in in vitro and in vivo experimental models of rodents transplanted with parental tumors or treated with carcinogens. We investigated the effects of dietary supplementation with annatto-T3 (90% delta-T3 and 10% gamma-T3) on the spontaneous development of mammary tumors in HER-2/neu transgenic mice. Underlying mechanisms of the antitumor effect were evaluated by studying apoptosis, senescent-like growth arrest, immune modulation, oxidative effect and the expression of HER-2/neu in tumoral mammary glands of transgenic mice and in vitro in human and mice tumor cell lines. Annatto-T3 supplementation delayed the development of mammary tumors, reducing the number and size of mammary tumor masses and those of lung metastases. In annatto-T3-supplemented mice, both apoptosis and senescent-like growth arrest of tumor cells were increased in mammary glands while no immune modulation was observed. In vitro, a dose-dependent inhibition of cell growth, increased apoptosis and senescent-like growth arrest and a time-dependent accumulation of reactive oxygen species were observed in tumor cells treated with annatto-T3 or purified delta-T3. Annatto-T3 reduced both HER-2/neu mRNA and p185(HER-2/neu) protein in tumors and in tumor cell lines. The results show that the antitumor effect of annatto-T3 supplementation in HER-2/neu transgenic mice is mainly related to the direct induction of oxidative stress, senescent-like growth arrest and apoptosis of tumor cells rather than to an immune modulation.
Tocotrienols promote apoptosis in human breast cancer cells by inducing poly(ADP-ribose) polymerase cleavage and inhibiting nuclear factor kappa-B activity
Loganathan, R.,Selvaduray, K. R.,Nesaretnam, K.,Radhakrishnan, A. K.
Cell Prolif 2013;46(2):203-13
OBJECTIVES: Tocotrienols and tocopherols are members of the vitamin E family, with similar structures; however, only tocotrienols have been reported to achieve potent anti-cancer effects. The study described here has evaluated anti-cancer activity of vitamin E to elucidate mechanisms of cell death, using human breast cancer cells. MATERIALS AND METHODS: Anti-cancer activity of a tocotrienol-rich fraction (TRF) and a tocotrienol-enriched fraction (TEF) isolated from palm oil, as well as pure vitamin E analogues (alpha-tocopherol, alpha-, delta- and gamma-tocotrienols) were studied using highly aggressive triple negative MDA-MB-231 cells and oestrogen-dependent MCF-7 cells, both of human breast cancer cell lines. Cell population growth was evaluated using a Coulter particle counter. Cell death mechanism, poly(ADP-ribose) polymerase cleavage and levels of NF-kappaB were determined using commercial ELISA kits. RESULTS: Tocotrienols exerted potent anti-proliferative effects on both types of cell by inducing apoptosis, the underlying mechanism of cell death being ascertained using respective IC50 concentrations of all test compounds. There was marked induction of apoptosis in both cell lines by tocotrienols compared to treatment with Paclitaxel, which was used as positive control. This activity was found to be associated with cleavage of poly(ADP-ribose) polymerase (a DNA repair protein), demonstrating involvement of the apoptotic cell death signalling pathway. Tocotrienols also inhibited expression of nuclear factor kappa-B (NF-kappaB), which in turn can increase sensitivity of cancer cells to apoptosis. CONCLUSION: Tocotrienols induced anti-proliferative and apoptotic effects in association with DNA fragmentation, poly(ADP-ribose) polymerase cleavage and NF-kappaB inhibition in the two human breast cancer cell lines.