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BACKGROUND&AIMS: Extracellular matrix deposition is key event for the development of bowel stenosis in Crohn’s disease patients. Transforming growth factor-β plays a key role in this process. We aimed at characterizing the effects of tocotrienol rich fraction on ECM proteins production and molecules that regulate the synthesis and degradation of extracellular matrix, matrix metalloproteinase-3 and tissue inhibitor of metalloproteinases-1, in human intestinal fibroblasts, and at elucidating whether the effects of tocotrienol rich fraction (TRF) are mediated through inhibition of TGF-β1.

METHODS: HIF were isolated from colonic or ileal tissue from Crohn’s disease patients and control subjects, and were treated with TRF from palm oil either alone or in combination with TGF-β1. Procollagen 1, procollagen 3, TIMP-1 and MMP-3 production, and Smad3 phosphorylation were analyzed by Western-blotting.

RESULTS: TRF significantly diminished procollagen 1 and 3 synthesis in HIF. Treatment of HIF with TRF increased MMP-3 production but did not modify TIMP-1. TGF-β1 induced Smad3 phosphorylation and enhanced procollagen 1 and 3 and TIMP-1 production. Pre-treatment of HIF with TRF prevented Smad3 phosphorylation and minimized the increase in collagen 1 and 3 production caused by TGF-β1.

CONCLUSIONS: TRF has anti-fibrogenic effects on HIF, decreasing ECM production and increasing its degradation. This effect is mediated, at least in part, by inhibition of TGF-β1

γ-Tocotrienol inhibits HGF-dependent mitogenesis and Met activation in highly malignant mammary tumour cells

N. M. Ayoub, S. V. Bachawal and P. W. Sylvester

Cell Prolif. 2011 Dec;44(6):516-26.

Objectives: Aberrant Met signalling is associated with aggressive cancer cell phenotypes. γ-tocotrienol displays potent anti-cancer activity that is associated with suppression of HER⁄ErbB receptor signalling. Experiments were conducted to investigate the effects of γ-tocotrienol treatment on HGF-dependent +SA mammary tumour cell proliferation, upon Met activation.

Materials and Methods: The +SA cells were maintained in serum-free defined media containing 10 ng ⁄ml HGF as the mitogen. Cell viability was determined using the MTT assay, western blot analysis was used to measure protein expression, and Met expression and activation were determined using immunofluorescent staining.

Results and Conclusions: Treatment with γ-tocotrienol or Met inhibitor, SU11274, significantly inhibited HGF-dependent +SA cell replication in a dose– responsive manner. Treatment with 4 lM c-tocotrienol reduced both total Met levels and HGF-induced Met autophosphorylation. In contrast, similar treatment with 5.5 lM SU11274 inhibited HGF-induced Met autophosphorylation, but had no effect on total Met levels. Combined treatment with subeffective doses of c-tocotrienol (2 lM) and SU11274 (3 lM) resulted in significant inhibition of +SA cell expansion compared to treatment with individual agents alone. These findings show, for the first time, the inhibitory effects of c-tocotrienol on Met expression and activation, and strongly suggest that c-tocotrienol treatment may provide significant health benefits in prevention and ⁄ or treatment of breast cancer, in women with deregulated HGF⁄Met signalling.

Vitamin E delta-tocotrienol augments the antitumor activity of gemcitabine and suppresses constitutive NF-kappaB activation in pancreatic cancer

Husain, K., Francois, R. A., Yamauchi, T., Perez, M., Sebti, S. M., Malafa, M. P.

Mol Cancer Ther. 2011 Dec;10(12):2363-72.

The NF-kappaB transcription factor functions as a crucial regulator of cell survival and chemoresistance in pancreatic cancer. Recent studies suggest that tocotrienols, which are the unsaturated forms of vitamin E, are a promising class of anticancer compounds that inhibit the growth and survival of many cancer cells, including pancreatic cancer. Here, we show that tocotrienols inhibited NF-kappaB activity and the survival of human pancreatic cancer cells in vitro and in vivo. Importantly, we found the bioactivity of the four natural tocotrienol compounds (alpha-, beta-, delta-, and gamma-tocotrienol) to be directly related to their ability to suppress NF-kappaB activity in vitro and in vivo. The most bioactive tocotrienol for pancreatic cancer, delta-tocotrienol, significantly enhanced the efficacy of gemcitabine to inhibit pancreatic cancer growth and survival in vitro and in vivo. Moreover, we found that delta-tocotrienol augmentation of gemcitabine activity in pancreatic cancer cells and tumors is associated with significant suppression of NF-kappaB activity and the expression of NF-kappaB transcriptional targets (Bcl-X(L), X-linked inhibitor of apoptosis, and survivin). Our study represents the first comprehensive preclinical evaluation of the activity of natural vitamin E compounds in pancreatic cancer. Given these results, we are conducting a phase I trial of delta-tocotrienol in patients with pancreatic cancer using pancreatic tumor cell survival and NF-kappaB signaling components as intermediate biomarkers. Our data also support future clinical investigation of delta-tocotrienol to augment gemcitabine activity in pancreatic cancer.

Byron J. Richards,

The kidneys may be the weak link in the chain when it comes to problems with blood pressure and blood sugar, which are at epidemic levels in America.  Nutrients that help protect the kidneys are likely to save untold health misery for potentially millions of people.  A new study by University of Arkansas researchers shows that gamma tocotrienol has potent kidney-protecting properties.

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Gamma-tocotrienol as an effective agent in targeting prostate cancer stem cell-like population

Luk, S. U.,Yap, W. N.,Chiu, Y. T.,Lee, D. T.,Ma, S.,Lee, T. K.,Vasireddy, R. S.,Wong, Y. C.,Ching, Y. P.,Nelson, C.,Yap, Y. L.,Ling, M. T.

Int J Cancer, 2011. 128(9):2182-91

Emerging evidence supports that prostate cancer originates from a rare subpopulation of cells, namely prostate cancer stem cells (CSCs). Conventional therapies for prostate cancer are believed to mainly target the majority of differentiated tumor cells but spare CSCs, which may account for the subsequent disease relapse after treatment. Therefore, successful elimination of CSCs may be an effective strategy to achieve complete remission from this disease. Gamma-tocotrienols (gamma-T3) is one of the vitamin-E constituents, which have been shown to have anticancer effects against a wide range of human cancers. Recently, we have reported that gamma-T3 treatment not only inhibits prostate cancer cell invasion but also sensitizes the cells to docetaxel-induced apoptosis, suggesting that gamma-T3 may be an effective therapeutic agent against advanced stage prostate cancer. Here, we demonstrate for the first time that gamma-T3 can downregulate the expression of prostate CSC markers (CD133/CD44) in androgen-independent prostate cancer cell lines (PC-3 and DU145), as evident from Western blotting analysis. Meanwhile, the spheroid formation ability of the prostate cancer cells was significantly hampered by gamma-T3 treatment. In addition, pretreatment of PC-3 cells with gamma-T3 was found to suppress tumor initiation ability of the cells. More importantly, although CD133-enriched PC-3 cells were highly resistant to docetaxel treatment, these cells were as sensitive to gamma-T3 treatment as the CD133-depleted population. Our data suggest that gamma-T3 may be an effective agent in targeting prostate CSCs, which may account for its anticancer and chemosensitizing effects reported in previous studies.

Protective effect of dietary tocotrienols against infection and inflammation-induced hyperlipidemia: An in vivo and in silico study

Salman Khan M, Akhtar S, Al-Sagair OA, Arif JM.

Phytother Res. 2011 Nov;25(11):1586-95. Epub 2011 Mar 11.

Currently used hypolipidemic drugs, Fluvastatin and Atorvastatin, act via inhibiting the rate-limiting enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase of the mevalonate pathway. The associated severe side-effects of these statins led us to explore the therapeutic potentials of naturally occurring Tocomin (mixture of dietary α-, β-, γ- and δ-tocotrienols). Tocomin (10 mg) was orally administered daily for 10 days before and 12 h after bacterial lipopolysaccharide (200 μg) or 24 h after zymosan (20 mg) or turpentine (0.5 mL) to Syrian hamsters. The data showed that Tocomin significantly reduced the levels of plasma and lipoprotein lipids, cholesterol, apoB, small dense (sd)-LDL as well as LDL in the hyperlipidemia-induced hamsters. Further, the mechanism of action of α-, β-, γ- and δ-tocotrienols was validated by docking studies with HMG-CoA reductase enzyme using the Molegro Virtual Docker. The inhibition of HMG-CoA reductase predicted in terms of MolDockScore and interaction energy suggest the comparative potential in the descending order: Atorvastatin > Fluvastatin ~ δ > γ > β > α. The results favor the daily intake of naturally occurring tocotrienols as dietary supplement in the prevention and treatment of infection/inflammation induced dyslipidemia compared with the hypolipidemic drugs.

Tocotrienol rich fraction (TRF) supplementation protects against oxidative DNA damage and improves cognitive functions in Wistar rats

Taridi NM, Yahaya MF, Teoh SL, Latiff AA, Ngah WZ, Das S, Mazlan M.

Clin Ter. 2011;162(2):93-8.

AIM: Oxidative stress is caused by imbalance between the productions of reactive oxygen species (ROS) and antioxidant defense mechanisms. Palm oil antioxidants such as tocotrienol rich fraction (TRF) is known to have neuroprotective effects on neurones by acting against free radical induced neuronal cell death. This study was undertaken to elucidate the effect of TRF on oxidative DNA damage and cognitive functions in experimental rats.

MATERIALS AND METHODS: A total of 20 male Wistar rats (aged 3 months) were divided into 2 groups: (i) control group fed with distilled water and (ii) experimental group fed with TRF (200 mg/ kg body weight) for 8 months. DNA damage was determined using Comet assay. Antioxidant enzymes like superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were assessed in the blood. The Morris Water Maze (MWM) test was used to evaluate the cognitive functions.

RESULTS: DNA damage was significantly reduced in the experimental group supplemented with TRF compared to the control group (p <0.05). In the group supplemented with TRF, the percentage of DNA damage was 2.87 ± 0.48% compared to 5.96 ± 0.43% in the control group. SOD, GPx, and CAT enzyme activities increased in experimental group. Results from MWM showed improvement in cognitive functions as determined by latency to target platform, swim path and average speed between TRF and control groups.

CONCLUSIONS: Continuous supplementation of TRF for 8 months reduced DNA damage and exhibited positive influence in spatial learning and memory.

Tocotrienol vitamin E protects against preclinical canine ischemic stroke by inducing arteriogenesis

Rink C, Christoforidis G, Khanna S, Peterson L, Patel Y, Khanna S, Abduljalil A, Irfanoglu O, Machiraju R, Bergdall VK, Sen CK

J Cereb Blood Flow Metab. 2011 Nov;31(11) Epub 2011 Jun 15.

Vitamin E consists of tocopherols and tocotrienols, in which α-tocotrienol is the most potent neuroprotective form that is also effective in protecting against stroke in rodents. As neuroprotective agents alone are insufficient to protect against stroke, we sought to test the effects of tocotrienol on the cerebrovascular circulation during ischemic stroke using a preclinical model that enables fluoroscopy-guided angiography. Mongrel canines (mean weight=26.3±3.2 kg) were supplemented with tocotrienol-enriched (TE) supplement (200 mg b.i.d, n=11) or vehicle placebo (n=9) for 10 weeks before inducing transient middle cerebral artery (MCA) occlusion. Magnetic resonance imaging was performed 1 hour and 24 hours post reperfusion to assess stroke-induced lesion volume. Tocotrienol-enriched supplementation significantly attenuated ischemic stroke-induced lesion volume (P<0.005). Furthermore, TE prevented loss of white matter fiber tract connectivity after stroke as evident by probabilistic tractography. Post hoc analysis of cerebral angiograms during MCA occlusion revealed that TE-supplemented canines had improved cerebrovascular collateral circulation to the ischemic MCA territory (P<0.05). Tocotrienol-enriched supplementation induced arteriogenic tissue inhibitor of metalloprotease 1 and subsequently attenuated the activity of matrix metalloproteinase-2. Outcomes of the current preclinical trial set the stage for a clinical trial testing the effects of TE in patients who have suffered from transient ischemic attack and are therefore at a high risk for stroke.

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Assessment of the protective effects of oral tocotrienols in arginine chronic-like pancreatitis

González AM, Garcia T, Samper E, Rickmann M, Vaquero EC, Molero X.

Am J Physiol Gastrointest Liver Physiol. 2011 Nov;301(5):G846-55. Epub 2011 Aug 18.

Tocotrienols exhibit anti-inflammatory properties over macrophages and promote cytotoxicity in activated pancreatic stellate cells, suggesting that they may limit chronic pancreatitis progression. We aimed to quantitate the effect of oral tocotrienols on a rat model of chronic pancreatic injury. Chronic-like pancreatitis was induced by repeated arginine pancreatitis. Palm oil tocotrienol-rich fraction (TRF) was given by gavage before and after pancreatitis inductions. Amylase and hydroxyproline were determined in pancreatic homogenates; collagen, fibronectin, α-smooth muscle actin (SMA), glial fibrillary acidic protein (GFAP), and phosphorylated Smad3 were assessed by Western blotting. Transforming growth factor (TGF)-β1 was measured in plasma. Morphological assessment included light microscopy, fibrosis area fraction, and collagen network fractal analysis. Arginine pancreatitis induced pancreatic atrophy and increased hydroxyproline that ameliorated after TRF. Arginine increased TGF-β1 (185 ± 40 vs. 15 ± 2 ng/ml; P <0.01) that was blunted by TRF (53 ± 19; P < 0.01). TRF reduced protease and Smad3 activation, collagen, and fibronectin. α-SMA increased and GFAP diminished in arginine pancreatitis, consistent with long-term stellate cell activation, and TRF reverted these changes to basal. Arginine pancreatitis increased fibrosis area fraction (4.5 ± 0.3% vs. 0.2 ± 0.2%), collagen network complexity (fractal dimension 1.52 ± 0.03 vs. 1.42 ± 0.01; P < 0.001), and inhomogeneity (lacunarity 0.63 ± 0.03 vs. 0.40 ± 0.02; P < 0.001), which were all reduced by TRF (1.3 ± 0.4%, 1.43 ± 0.02%, and 0.51 ± 0.03%, respectively; P < 0.01). Best correlation coefficients were obtained when comparing fibrosis area fraction with lacunarity (r = 0.88) and both parameters with pancreatic weight (r = -0.91 and -0.79, respectively). TRF administered only before pancreatitis best, but not fully, recapitulated the beneficial effects of TRF. Tocotrienols improve quantitative measures of chronic pancreatic damage. They may be of benefit in human chronic pancreatitis.

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Tocotrienols, like tocopherols, are members of the vitamin E family. While tocopherols (T) have been studied intensively, only recently havetocotrienols (T3) received increased attention due to their special health benefits. However, these positive attributes of T3 are probably lost as a result of degradation during food storage and processing, and there is little information about their oxidation products. Of particular interest are the oxidation products of α-tocotrienol (α-T3) as this is the least thermostable T3 isomer with the highest rate of degradation. The objective of this study was therefore to develop a reliable method for the determination of the most important oxidation products of α-T3 along with other tocochromanol isomers. We developed a high-performance liquid chromatography method with diode array detection, fluorescence detection, and a particle beam interface electron impact mass spectroscopy in order to separate the most important oxidation products of α-T3 (α-T3 spirodimers/spirotrimers, α-tocotrienoldihydroxy dimer, 7-formyl-β-tocotrienol (7-FβT3), 5-formyl-γ-tocotrienol (5-FγT3), α-tocotrienolquinone (α-T3Q), and α-T3Q dimers and α-tocotrienolquinone epoxides (α-T3QE)) from eight tocochromanol isomers. Furthermore, we sought to identify the as yet unknown oxidation products 5-FγT3, 7-FβT3, α-T3Q-dimer, and α-T3QE. Of these, 5-FγT3 was fully characterized by Fourier transform infrared spectroscopy and (1)H and (13)C nuclear magnetic resonance spectroscopy.