Although cell-based studies have shown that gamma-tocotrienol (gammaTE) exhibits stronger anticancer activities than other forms of vitamin E including gamma-tocopherol (gammaT), the molecular bases underlying gammaTE-exerted effects remains to be elucidated. Here we showed that gammaTE treatment promoted apoptosis, necrosis and autophagy in human prostate PC-3 and LNCaP cancer cells. In search of potential mechanisms of gammaTE-provoked effects, we found that gammaTE treatment led to marked increase of intracellular dihydroceramide and dihydrosphingosine, the sphingolipid intermediates in de novo sphingolipid synthesis pathway but had no effects on ceramide or sphingosine. The elevation of these sphingolipids by gammaTE preceded or coincided with biochemical and morphological signs of cell death and was much more pronounced than that induced by gammaT, which accompanied with much higher cellular uptake of gammaTE than gammaT. The importance of sphingolipid accumulation in gammaTE-caused fatality was underscored by the observation that dihydrosphingosine and dihydroceramide potently reduced the viability of both prostate cell lines and LNCaP cells, respectively. In addition, myriosin, a specific inhibitor of de novo sphingolipid synthesis, counteracted gammaTE-induced cell death. In agreement with these cell-based studies, gammaTE inhibited LNCaP xenograft growth by 53% (p < 0.05), compared to 33% (p = 0.07) by gammaT, in nude mice. These findings provide a molecular basis of gammaTE-stimulated cancer cell death and support the notion that elevation of intracellular dihydroceramide and dihydrosphingosine is likely a novel anticancer mechanism.
Publications
Tocotrienols prevent hydrogen peroxide-induced axon and dendrite degeneration in cerebellar granule cells
Fukui K, Ushiki K, Takatsu H, Koike T, Urano S.
Free Radic Res. 2012 Feb;46(2):184-93.
It is well known that reactive oxygen species (ROS) attack several living tissues and increase the risk of development and progression of serious diseases. In neuronal level, ROS induce cell death in concentration-dependent fashion. However, little is known about the mechanisms of neuronal changes by ROS prior to induction of cell death. Here we found that treatment of cerebellar granule neurons (CGCs) with 0.5 μM hydrogen peroxide induced axonal injury, but not cell death. The number of dendrites remarkably decreased in hydrogen peroxide-treated CGCs, and extensive beading was observed on survival dendrites. In addition, an abnormal band of the original collapsin response mediator protein (CRMP)-2 was detected by Western blotting in hydrogen peroxide-treated CGCs. Treatment with each tocotrienol isoform prevented axonal and dendrite degeneration and induction of the abnormal band of the original band of CRMP-2 in hydrogen peroxide-treated CGCs. These results indicate that treatment with tocotrienols may therefore be neuroprotective in the presence of hydrogen peroxide by preventing changes to the CRMP-2 that occur before neuron death.
Metabolism of tocotrienols in animals and synergistic inhibitory actions of tocotrienols with atorvastatin in cancer cells
Yang Z, Lee MJ, Zhao Y, Yang CS.
Genes Nutr. 2012 Jan;7(1):11-8. Epub 2011 May 18.
Tocotrienols (T3s), members of the vitamin E family, exhibit potent anti-cancer, anti-oxidative, anti-inflammatory, and some other biological activities. To better understand the bioavailability and metabolism of T3s, T3s and their metabolites were identified in urine and fecal samples from mice on diet supplemented with mixed T3s using HPLC/electrochemical detection and liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS). Whereas the short-chain metabolites carboxyethyl hydroxychromans (CEHCs) and carboxymethylbutyl hydroxychromans (CMBHCs) were the major metabolites of T3s, several new metabolites with double bonds were also identified. Similar to tocopherols, the majority of T3 metabolites were excreted as sulfate/glucuronide conjugates in mouse urine. The distribution of γ- and δ-T3 and γ-T3 metabolites were also determined in different organs as well as in urine and fecal samples from mice on diets supplemented with corresponding T3s. The synergistic anti-cancer actions of γ-T3 and atorvastatin (ATST) were studied in HT29 and HCT116 colon cancer cell lines. The combination greatly potentiated the ability of each individual agent to inhibit cancer cell growth and to induce cell cycle arrest and apoptosis. The triple combination of γ-T3, ATST, and celecoxib exhibitedsynergistic actions when compared with any double combination plus the third agent. Mechanistic studies revealed that the synergistic actions of γ-T3 and ATST could be attributed to their mediation of 3-hydroxy-3-methyl-glutaryl-CoA reductase, and the subsequent inhibition of protein geranylgeranylation. It remains to be determined whether such a synergy occurs in vivo.
Tocotrienols fight cancer by targeting multiple cell signaling pathways
Kannappan R, Gupta SC, Kim JH, Aggarwal BB.
Genes Nutr. 2012 Jan;7(1):43-52. Epub 2011 Apr 9.
Cancer cells are distinguished by several distinct characteristics, such as self-sufficiency in growth signal, resistance to growth inhibition, limitless replicative potential, evasion of apoptosis, sustained angiogenesis, and tissue invasion and metastasis. Tumor cells acquire these properties due to the dysregulation of multiple genes and associated cell signaling pathways, most of which are linked to inflammation. For that reason, rationally designed drugs that target a single gene product are unlikely to be of use in preventing or treating cancer. Moreover, targeted drugs can cause serious and even life-threatening side effects. Therefore, there is an urgent need for safe and effective promiscuous (multitargeted) drugs. “Mother Nature” produces numerous such compounds that regulate multiple cell signaling pathways, are cost effective, exhibit low toxicity, and are readily available. One among these is tocotrienol, a member of the vitamin E family, which has exhibited anticancer properties. This review summarizes data from in vitro and in vivo studies of the effects of tocotrienol on nuclear factor-κB, signal transducer and activator of transcription (STAT) 3, death receptors, apoptosis, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), hypoxia-inducible factor (HIF) 1, growth factor receptor kinases, and angiogenic pathways.
Gamma-tocotrienol induced apoptosis is associated with unfolded protein response in human breast cancer cells
Patacsil D, Tran AT, Cho YS, Suy S, Saenz F, Malyukova I, Ressom H, Collins SP, Clarke R, Kumar D.
J Nutr Biochem. 2012 Jan;23(1):93-100. Epub 2011 Mar 22.
Gamma-tocotrienol (γ-T3) is a member of the vitamin E family. Tocotrienols (T3s) are powerful antioxidants and possess anticancer, neuroprotective and cholesterol-lowering properties. Tocotrienols inhibit the growth of various cancer cell lines without affecting normal cells. Less is known about the exact mechanisms of action of T3s on cell death and other growth inhibitory pathways. In the present study, we demonstrate that γ-T3 induces apoptosis in MDA-MB 231 and MCF-7 breast cancer cells as evident by PARP cleavage and caspase-7 activation. Gene expression analysis of MCF-7 cells treated with γ-T3 revealed alterations in the expression of multiple genes involved in cell growth and proliferation, cell death, cell cycle, cellular development, cellular movement and gene expression. Further analysis of differentially modulated genes using Ingenuity Pathway Analysis software suggested modulation of canonical signal transduction or metabolic pathways such as NRF-2-mediated oxidative stress response, TGF-β signaling and endoplasmic reticulum (ER) stress response. Analysis of ER-stress-related proteins in MCF-7 and MDA-MB 231 cells treated with γ-T3 demonstrated activation of PERK and pIRE1α pathway to induce ER stress. Activating transcription factor 3 (ATF3) was identified as the most up-regulated gene (16.8-fold) in response to γ-T3. Activating transcription factor 3 knockdown using siRNA suggested an essential role of ATF3 in γ-T3-induced apoptosis. In summary, we demonstrate that γ-T3 modulates ER stress signaling and have identified ATF3 as a molecular target for γ-T3 in breast cancer cells.
Why tocotrienols work better:Insights into the in vitro anti-cancer mechanism of vitamin E
Viola V, Pilolli F, Piroddi M, Pierpaoli E, Orlando F, Provinciali M, Betti M, Mazzini F, Galli F.
Genes Nutr. 2012 Jan;7(1):29-41. Epub 2011 Apr 20.
The selective constraint of liver uptake and the sustained metabolism of tocotrienols (T3) demonstrate the need for a prompt detoxification of this class of lipophilic vitamers, and thus the potential for cytotoxic effects in hepatic and extra-hepatic tissues. Hypomethylated (γ and δ) forms of T3 show the highest in vitro and in vivo metabolism and are also the most potent natural xenobiotics of the entire vitamin E family of compounds. These stimulate a stress response with the induction of detoxification and antioxidant genes. Depending on the intensity of this response, these genes may confer cell protection or alternatively they stimulate a senescence-like phenotype with cell cycle inhibition or even mitochondrial toxicity and apoptosis. In cancer cells, the uptake rate and thus the cell content of these vitamers is again higher for the hypomethylated forms, and it is the critical factor that drives the dichotomy between protection and toxicity responses to different T3 forms and doses. These aspects suggest the potential for marked biological activity of hypomethylated “highly metabolized” T3 that may result in cytoprotection and cancer prevention or even chemotherapeutic effects. Cytotoxicity and metabolism of hypomethylated T3 have been extensively investigated in vitro using different cell model systems that will be discussed in this review paper as regard molecular mechanisms and possible relevance in cancer therapy.
Mechanisms underlying the radioprotective properties of γ-tocotrienol: Comparative gene expression profiling in tocol-treated endothelial cells
Berbée M, Fu Q, Boerma M, Sree Kumar K, Loose DS, Hauer-Jensen M.
Genes Nutr. 2012 Jan;7(1):75-81. Epub 2011 Apr 24.
Among the eight naturally occurring vitamin E analogs, γ-tocotrienol (GT3) is a particularly potent radioprophylactic agent in vivo. Moreover, GT3 protects endothelial cells from radiation injury not only by virtue of its antioxidant properties but also by inhibition of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and by improving the availability of the nitric oxide synthase cofactor tetrahydrobiopterin. Nevertheless, the precise mechanisms underlying the superior radioprotective properties of GT3 compared with other tocols are not known. This study, therefore, examined the differences in gene expression profiles between GT3 and its tocopherol counterpart, γ-tocopherol, as well as between GT3 and α-tocopherol in human endothelial cells. Cells were treated with vehicle or the appropriate tocol for 24 h, after which total RNA was isolated and genome-wide gene expression profiles were obtained using the Illumina platform. GT3 was far more potent in inducing gene-expression changes than α-tocopherol or γ-tocopherol. In particular, GT3 induced multiple changes in pathways known to be of importance in the cellular response to radiation exposure. Affected GO functional clusters included response to oxidative stress, response to DNA damage stimuli, cell cycle phase, regulation of cell death, regulation of cell proliferation, hematopoiesis, and blood vessel development. These results form the basis for further studies to determine the exact importance of differentially affected GO functional clusters in endothelial radioprotection by GT3.
Synergistic anticancer effects of combined γ-tocotrienol with statin or receptor tyrosine kinase inhibitor treatment
Sylvester PW.
Genes Nutr. 2012 Jan;7(1):63-74. Epub 2011 May 1.
Systemic chemotherapy is the only current method of treatment that provides some chance for long-term survival in patients with advanced or metastatic cancer. γ-Tocotrienol is a natural form of vitamin E found in high concentrations in palm oil and displays potent anticancer effects, but limited absorption and transport of by the body has made it difficult to obtain and sustain therapeutic levels in the blood and target tissues. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase and are an example of a promising cancer chemotherapeutic agent whose clinical usefulness has been limited due to high-dose toxicity. Similarly, erlotinib and gefitinib are anticancer agents that inhibit the activation of individual HER/ErbB receptor subtypes, but have shown limited clinical success because of heterodimerization between different EGF receptor family members that can rescue cancer cells from agents directed against a single receptor subtype. Recent studies have investigated the anticancer effectiveness of low-dose treatment of various statins or EGF receptor inhibitors alone and in combination with γ-tocotrienol on highly malignant +SA mouse mammary epithelial cells in vitro. Combined treatment with subeffective doses of γ-tocotrienol with these other chemotherapeutic agents resulted in a synergistic inhibition of +SA cell growth and viability. These findings strongly suggest that combined treatment of γ-tocotrienol with other anticancer agents may not only provide an enhanced therapeutic response but also provide a means to avoid the toxicity, low bioavailability, or limited therapeutic action associated with high-dose monotherapy.
Tocotrienols confer resistance to ischemia in hypercholesterolemic hearts: Insight with genomics
Das S, Mukherjee S, Lekli I, Gurusamy N, Bardhan J, Raychoudhury U, Chakravarty R, Banerji S, Knowlton AA, Das DK.
Mol Cell Biochem. 2012 Jan;360(1-2):35-45. Epub 2011 Sep 15.
Most clinical trials with vitamin E could not lower cholesterol and thus, have been deemed unsuccessful. Recently, tocotrienols, isomers of vitamin E have been found to lower LDL levels. To explore if tocotrienols could be the drug target for vitamin E, rabbits were kept on cholesterol diet for 60 days supplemented with tocotrienol-α, tocotrienol-δ, and tocotrienol-γ for the last 30 days. The serum cholesterol levels (in mmol/l) were 24.4 (tocotrienol-α), 34.9 (tocotrienol-δ), 19.8 (tocotrienol-γ) vs. 39.7 (control). Left ventricular function including aortic flow and developed pressure exhibited significantly improved recovery with tocotrienol-γ and -α, but not with tocotrienol-δ. The myocardial infarct size showed a similar pattern: 33% (tocotrienol-α), 23% (tocotrienol-γ), and 47% (tocotrienol-δ). To examine the molecular mechanisms of cardioprotective effects, gene expression profile was determined using Atlas 1.2/1.2II followed by determination of gene profiles using PedQuest 8.3 software. Based on genomic profiles, the following cholesterol-related proteins were examined: FABP, TGF-β (cholesterol suppresses TGF-β), ET-1 (increased by hypercholesterolemia), SPOT 14 (linked with hypercholesterolemia), and matrix metalloproteinase (MMP) 2 and MMP9 (cholesterol regulates MMP2 and MMP9 expression) in the heart. Consistent with the cardioprotective effects of tocotrienol-α and -γ, these two isomers reduced ET-1, decreased MMP2 and MM9, increased TGF-β and reduced SPOT 14, while tocotrienol-δ had no effects. The results of the present study demonstrate that the two isomers of tocotrienols, α and γ, render the hypercholesterolemic hearts resistant to ischemic reperfusion injury by lowering several hypercholesterolemic proteins including MMP2, MMP9, ET-1, and SPOT 14 and upregulating TGF-β.
Alpha-tocotrienol is the most abundant tocotrienol isomer circulated in plasma and lipoproteins after postprandial tocotrienol-rich vitamin E supplementation
Fairus S, Nor RM, Cheng HM, Sundram K.
Nutr J. 2012 Jan 17;11:5.
Introduction: Tocotrienols (T3) and tocopherols (T), both members of the natural vitamin E family have unique biological functions in humans. T3 are detected in circulating human plasma and lipoproteins, although at concentrations significantly lower than α-tocopherol (α-T). T3, especially α-T3 is known to be neuropotective at nanomolar concentrations and this study evaluated the postprandial fate of T3 and α-T in plasma and lipoproteins.