Anti-angiogenic therapy is widely being used to halt tumour angiogenesis. In this study, the anti-angiogenic activity of palm tocotrienol-rich fraction (TRF) and its individual components (γ- and δ-tocotrienol) were first investigated in vitro in human umbilical vein endothelial cells (HUVEC) and 4T1 mouse mammary cancer cells. Results showed reduced levels of Interkeukin (IL)-8 and IL-6, two pro-angiogenic cytokines in HUVEC treated with palm tocotrienols compared with α-tocopherol (α-T) and control cells (P < 0.05). The production of IL-8 and IL-6 was lowest in δ-tocotrienol (δ-T3)-treated cells followed by γ-tocotrienol (γ-T3) and TRF. There was significant (P < 0.05) reduction in IL-8 and vascular endothelial growth factor (VEGF) production in 4T1 cells treated with TRF or δ-T3. There was decreased expression of VEGF and its receptors; VEGF-R1 (fms-like tyrosine kinase, Flt-1) and VEGF-R2 (Kinase-insert-domain-containing receptor, KDR/Flk-2) in tumour tissues excised from mice supplemented with TRF were observed. There was also decreased expression of VEGF-R2 in lung tissues of mice supplemented with TRF. These observations correlate with the smaller tumour size recorded in the tocotrienol-treated mice. This study confirms previous observations that palm tocotrienols exhibit anti-angiogenic properties that may inhibit tumour progression.
Publications
Tocotrienols and breast cancer: The evidence to date
Kalanithi Nesaretnam, Puvaneswari Meganathan, Sheela Devi Veerasenan, Kanga Rani Selvaduray
Genes Nutr. 2012 Jan;7(1):3-9.
Breast cancer is the second most frequent cancer affecting women worldwide after lung cancer. The toxicity factor associated with synthetic drugs has turned the attention toward natural compounds as the primary focus of interest as anticancer agents. Vitamin E derivatives consisting of the well-established tocopherols and their analogs namely tocotrienols have been extensively studied due to their remarkable biological properties. While tocopherols have failed to offer protection, tocotrienols, in particular, a-, d-, and c-tocotrienols alone and in combination have demonstrated anticancer properties. The discovery of the antiangiogenic, antiproliferative, and apoptotic effects of tocotrienols, as well as their role as an inducer of immunological functions, not only reveals a new horizon as a potent antitumor agent but also reinforces the notion that tocotrienols are indeed more than antioxidants. On the basis of a transcriptomic platform, we have recently demonstrated a novel mechanism for tocotrienol activity that involves estrogen receptor (ER) signaling. In silico simulations and in vitro binding analyses indicate a high affinity of specific forms of tocotrienols for ERb, but not for ERa. Moreover, we have demonstrated that specific tocotrienols increase ERb translocation into the nucleus which, in turn, activates the expression of estrogenresponsive genes (MIC-1, EGR-1 and Cathepsin D) in breast cancer cells only expressing ERb cells (MDA-MB-231) and in cells expressing both ER isoforms (MCF-7). The binding of specific tocotrienol forms to ERb is associated with the alteration of cell morphology, caspase-3 activation, DNA fragmentation, and apoptosis. Furthermore, a recently concluded clinical trial seems to suggest that tocotrienols in combination with tamoxifen may have the potential to extend breast cancer-specific survival.
γ-Tocotrienol does not substantially protect DS neurons from hydrogen peroxide-induced oxidative injury
Then SM, Sanfeliu C, Top GM, Wan Ngah WZ, Mazlan M.
Nutr Metab (Lond). 2012 Jan 5;9:1.
Background: Down syndrome (DS) neurons are more susceptible to oxidative stress and previous studies have shown that vitamin E was able to reduce oxidative stress and improve DS neurons’ viability. Therefore, this study was done to investigate the protective role of γ-tocotrienol (γT3) in DS neurons from hydrogen peroxide (H2O2) -induced oxidative stress. The pro-apoptosis tendency of γT3 was compared to α-tocopherol (αT) in non-stress condition as well.
Methods: Primary culture of DS and euploid neurons were divided into six groups of treatment: control, H2O2, γT3 pre-treatment with H2O2, γT3 only, αT pre-treatment with H2O2 and αT only. The treatments were assessed by MTS assay and apoptosis assay by single-stranded DNA (ssDNA) apoptosis ELISA assay, Hoechst and Neu-N immunofluorescence staining. The cellular uptake of γT3 and αT was determined by HPLC while protein expressions were determined by Western blot. Comparison between groups was made by the Student’s t test, one-way ANOVA and Bonferroni adjustment as well as two-way ANOVA for multiple comparisons.
Results: One day incubation of γT3 was able to reduced apoptosis of DS neurons by 10%, however γT3 was cytotoxic at longer incubation period (14 days) and at concentrations ≥ 100 μM. Pre-treatment of αT and γT3 only attenuate apoptosis and increase cell viability in H2O2-treated DS and euploid neurons by 10% in which the effects were minimal to maintain most of the DS cells’ morphology. γT3 act as a free radical scavenger by reducing ROS generated by H2O2. In untreated controls, DS neurons showed lower Bcl-2/Bax ratio and p53 expression compared to normal neurons, while cPKC and PKC-δ expressions were higher in DS neurons. On the other hand, pre-treatment of γT3 in H2O2-treated DS neurons have reduced Bcl-2/Bax ratio, which was not shown in euploid neurons. This suggests that pre-treatment of γT3 did not promote DS cell survival. Meanwhile γT3 and αT treatments without H2O2 as well as pre-treatment of γT3 and αT induced changes in cPKC and PKC-δ expression in DS neurons suggesting interaction of γT3 and αT with PKC activity.
Conclusion: Our study suggests that γT3 pre-treatment are not sufficient to protect DS neurons from H2O2-induced oxidative assault, instead induced the apoptosis process.
Do tocotrienols have potential as neuroprotective dietary factors?
Frank J, Chin XW, Schrader C, Eckert GP, Rimbach G.
Ageing Res Rev. 2012 Jan;11(1):163-80
Tocotrienols (T(3)) belong to the family of vitamin E compounds (α-, β-, γ-, δ-tocopherols and -tocotrienols) and have unique biological properties that make them potential neuroprotective dietary factors. In addition to their antioxidant activity, T(3) at micromolar concentrations exert cholesterol-lowering activities in cells, animal models and some, but not all, human studies by means of inhibition of the activity of the rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase. At lower concentrations (∼10nmol/L), T(3) modulate signalling pathways involved in neuronal cell death in cell culture experiments. Targets of T(3) include prenyl transferases, non-receptor tyrosine kinase, phospholipase A(2), 12-lipoxygenase, cyclooxygenase-2, and nuclear factor κB. The low bioavailability and rapid excretion of T(3) represents a major hurdle in their preventive use. Fasting plasma concentrations, even after supplementation with high doses, are below 1μmol/L. T(3) bioavailability may be enhanced by ingestion with a high-fat meal, self-emulsifying drug delivery systems, or phytochemicals that inhibit T(3) metabolism and excretion. T(3) have no known adverse effects when consumed as part of a normal diet and the studies reviewed here support the notion that they may have potential as neuroprotective agents. However, experiments in relevant animal models and randomised human intervention trials addressing the neuroprotection mediated by T(3) are scarce and, thus, highly warranted.
γ-Tocotrienol inhibits angiogenesis of human umbilical vein endothelial cell induced by cancer cell
Li Y, Sun WG, Liu HK, Qi GY, Wang Q, Sun XR, Chen BQ, Liu JR.
J Nutr Biochem. 2011 Dec;22(12):1127-36. Epub 2011 Feb 2.
Antiangiogenic therapy mediated by food components is an established strategy for cancer chemoprevention. Growth factors play critical roles in tumor angiogenesis. A conditioned medium containing growth factors from human gastric adenocarcinoma SGC-7901 cell conditioned medium was used as an angiogenic stimulus in this study. The purpose of this study was to evaluate the inhibitory effect and possible mechanism of γ-tocotrienolon tumor angiogenesis. The results showed that γ-tocotrienol (10-40 μmol/L) significantly suppressed proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) induced by SGC-7901 cell conditioned medium in a dose-dependent manner. γ-Tocotrienol (800-1200 μg/egg) also inhibited new blood vessel formation on the growing chick embryo chorioallantoic membrane in a dose-dependent manner. Moreover, the inhibitory effects of γ-tocotrienol on HUVECs were correlated with inducing the apoptosis and arresting cell cycle at the G(0)/G(1) phase at a dose of 40 μmol/L γ-tocotrienol. In addition, γ-tocotrienol inhibited angiogenesis in HUVECs by down-regulation of β-catenin, cyclin D1, CD44, phospho-VEGFR-2 and MMP-9. The antiangiogenic effects of γ-tocotrienol on HUVECs may be attributable to regulation of Wnt signaling by decreasing β-catenin expression. Thus, our results suggest that γ-tocotrienol has a potential chemopreventive agent via antiangiogenesis.
Inhibition of nitric oxide in LPS-stimulated macrophages of young and senescent mice by δ-tocotrienol and quercetin
Qureshi AA, Tan X, Reis JC, Badr MZ, Papasian CJ, Morrison DC, Qureshi N.
Lipids Health Dis. 2011 Dec 20;10:239.
Background: Changes in immune function believed to contribute to a variety of age-related diseases have been associated with increased production of nitric oxide (NO). We have recently reported that proteasome inhibitors (dexamethasone, mevinolin, quercetin, δ-tocotrienol, and riboflavin) can inhibit lipopolysaccharide (LPS)-induced NO production in vitro by RAW 264.7 cells and by thioglycolate-elicited peritoneal macrophages derived from four strains of mice (C57BL/6, BALB/c, LMP7/MECL-1(-/-) and PPAR-α(-/-) knockout mice). The present study was carried out in order to further explore the potential effects of diet supplementation with naturally-occurring inhibitors (δ-tocotrienol and quercetin) on LPS-stimulated production of NO, TNF-α, and other pro-inflammatory cytokines involved in the ageing process. Young (4-week-old) and senescent mice (42-week old) were fed control diet with or without quercetin (100 ppm), δ-tocotrienol (100 ppm), or dexamethasone (10 ppm; included as positive control for suppression of inflammation) for 4 weeks. At the end of feeding period, thioglycolate-elicited peritoneal macrophages were collected, stimulated with LPS, LPS plus interferon-β (IFN-β), or LPS plus interferon-γ (IFN-γ), and inflammatory responses assessed as measured by production of NO and TNF-α, mRNA reduction for TNF-α, and iNOS genes, and microarray analysis.
Results: Thioglycolate-elicited peritoneal macrophages prepared after four weeks of feeding, and then challenged with LPS (10 ng or 100 ng) resulted in increases of 55% and 73%, respectively in the production of NO of 46-week-old compared to 8-week-old mice fed control diet alone (respective control groups), without affecting the secretion of TNF-α among these two groups. However, macrophages obtained after feeding with quercetin, δ-tocotrienol, and dexamethasone significantly inhibited (30% to 60%; P < 0.02) the LPS-stimulated NO production, compared to respective control groups. There was a 2-fold increase in the production of NO, when LPS-stimulated macrophages of quercetin, δ-tocotrienol, or dexamethasone were also treated with IFN-β or IFN-γ compared to respective control groups. We also demonstrated that NO levels and iNOS mRNA expression levels were significantly higher in LPS-stimulated macrophages from senescent (0.69 vs 0.41; P < 0.05), compared to young mice. In contrast, age did not appear to impact levels of TNF-α protein or mRNA expression levels (0.38 vs 0.35) in LPS-stimulated macrophages. The histological analyses of livers of control groups showed lesions of peliosis and microvesicular steatosis, and treated groups showed Councilman body, and small or large lymphoplasmacytic clusters.
Conclusions: The present results demonstrated that quercetin and δ-tocotrienols inhibit the LPS-induced NO production in vivo. The microarray DNA analyses, followed by pathway analyses indicated that quercetin or δ-tocotrienol inhibit several LPS-induced expression of several ageing and pro-inflammatory genes (IL-1β, IL-1α, IL-6, TNF-α, IL-12, iNOS, VCAM1, ICAM1, COX2, IL-1RA, TRAF1 and CD40). The NF-κB pathway regulates the production of NO and inhibits the pro-inflammatory cytokines involved in normal and ageing process. These ex vivo results confirmed the earlier in vitro findings. The present findings of inhibition of NO production by quercetin and δ-tocotrienol may be of clinical significance treating several inflammatory diseases, including ageing process.
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.
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.