A comparative study investigated four tocopherols, four tocotrienols, and alpha-tocopheryl acetate on their antioxidative activities in five different popular assays, which were adapted to non-polar antioxidants. alpha-Tocopherol, used as calibration standard, showed the highest ferric reducing antioxidant power. Greater ring methyl substitution not only led to an increase of scavenging activity against the stable 2,2-diphenyl-1-picrylhydrazyl radical, but also to a decrease in oxygen radical absorbance capacity. Regarding alpha-tocopherol equivalent antioxidant capacity no significant differences in the antioxidant activity of all vitamin E isoforms were found. In contrast, a significantly lower peroxyl radical-scavenging activity of alpha-tocochromanols was determined in a chemiluminescence assay. Except oxygen radical absorbance capacity, no significant differences of the antioxidant activity related to the side chain could be detected. The data show that the reducing ability and radical chain-breaking activity of the several vitamin E forms depends on the circumstances under which the assays are performed. In our opinion, the used lipophilic methods can be useful for estimating the antioxidant activity of strong non-polar antioxidants, e.g. carotenoids, too. Furthermore, we could show a significant correlation between the total tocopherol content in human plasma and the lipophilic antioxidant capacity measured by alpha-tocopherol equivalent antioxidant capacity and 2,2-diphenyl-1-picrylhydrazyl.
Publications
Tocotrienols activity in MCF-7 breast cancer cells: Involvement of ERbeta signal transduction
Comitato R, Leoni G, Canali R, Ambra R, Nesaretnam K, Virgili F.
Mol Nutr Food Res. 2010 May;54(5):669-78.
The term Vitamin E is utilized to describe eight molecules, subdivided into two groups, tocopherols and tocotrienols (TTs). It has been shown that specific TTs affect the growth of several lines of tumour cells, and that this activity is not shared by tocopherols. In agreement with these observations, a TTs-rich fraction from palm oil (PTRF) was reported to inhibit proliferation and induce apoptosis in several cancer cells. However, the molecular mechanism involved in TTs activity is still unclear. We have recently proposed that TTs pro-apoptotic activity involves estrogen receptor beta (ERbeta) signalling. In this study, we report that, in MCF-7 breast cancer cell, expressing both ERalpha and ERbeta, PTRF treatment increases ERbeta nuclear translocation, as demonstrated by immunofluorescence experiments and significantly inhibits ERalpha expression (-458.91-fold of change) and complete disappearing of the protein from the nucleus. Moreover, PTRF treatment induces ER-dependent genes expression (macrophage inhibitory cytokine-1, early growth response-1 and Cathepsin D) which is inhibited by the ER inhibitor, ICI 182.780, and induces DNA fragmentation. Finally, cDNA-array experiments suggest that the activation of specific pathways in cells treated with gamma-TT with respect to alpha-TT. Our data suggest a novel potential molecular mechanism for TTs activity.
Id1, inhibitor of differentiation, is a key protein mediating anti-tumor responses of gamma-tocotrienol in breast cancer cells
Yap WN, Zaiden N, Tan YL, Ngoh CP, Zhang XW, Wong YC, Ling MT, Yap YL
Cancer Lett. 2010 May 28;291(2):187-99. Epub 2009 Nov 18.
Gamma-tocotrienol has demonstrated anti-proliferative effect on breast cancer (BCa) cells, but mechanisms involved are largely unknown. This study aimed at deciphering the molecular pathways responsible for its activity. Our results showed that treatment of BCa cells with gamma-tocotrienol resulted in induction of apoptosis as evidenced by activation of pro-caspases, accumulation of sub-G1 cells and DNA fragmentations. Examination of the pro-survival genes revealed that the gamma-tocotrienol-induced cell death was associated with suppression of Id1 and NF-kappaB through modulation of their upstream regulators (Src, Smad1/5/8, Fak and LOX). Meanwhile, gamma-tocotrienol treatment also resulted in the induction of JNK signaling pathway and inhibition of JNK activity by specific inhibitor partially blocked the effect of gamma-tocotrienol. Furthermore, synergistic effect was observed when cells were co-treated with gamma-tocotrienol and Docetaxel. Interestingly, in cells that treated with gamma-tocotrienol, alpha-tocopherol or beta-aminoproprionitrile were found to partially restore Id1 expression. Meanwhile, this restoration of Id1 was found to protect the cells from gamma-tocotrienol induced apoptosis. Consistent outcome was observed in cells ectopically transfected with the Id-1 gene. Our results suggested that the anti-proliferative and chemosensitization effect of gamma-tocotrienol on BCa cells may be mediated through downregulation of Id1 protein.
Cancer-preventive activities of tocopherols and tocotrienols
Ju J, Picinich SC, Yang Z, Zhao Y, Suh N, Kong AN, Yang CS.
Carcinogenesis. 2010 Apr;31(4):533-42. Epub 2009 Sep 11.
The cancer-preventive activity of vitamin E has been studied. Whereas some epidemiological studies have suggested a protective effect of vitamin E against cancer formation, many large-scale intervention studies with alpha-tocopherol (usually large doses) have not demonstrated a cancer-preventive effect. Studies on alpha-tocopherol in animal models also have not demonstrated robust cancer prevention effects. One possible explanation for the lack of demonstrable cancer-preventive effects is that high doses of alpha-tocopherol decrease the blood and tissue levels of delta-tocopherols. It has been suggested that gamma-tocopherol, due to its strong anti-inflammatory and other activities, may be the more effective form of vitamin E in cancer prevention. Our recent results have demonstrated that a gamma-tocopherol-rich mixture of tocopherols inhibits colon, prostate, mammary and lung tumorigenesis in animal models, suggesting that this mixture may have a high potential for applications in the prevention of human cancer. In this review, we discuss biochemical properties of tocopherols, results of possible cancer-preventive effects in humans and animal models and possible mechanisms involved in the inhibition of carcinogenesis. Based on this information, we propose that a gamma-tocopherol-rich mixture of tocopherols is a very promising cancer-preventive agent and warrants extensive future research.
Preparation, characterization, and anticancer effects of simvastatin-tocotrienol lipid nanoparticles
Ali H, Shirode AB, Sylvester PW, Nazzal S.
Int J Pharm. 2010 Apr 15;389(1-2):223-31. Epub 2010 Feb 1.
Previously it was shown that combined low dose treatment of tocotrienols and statins synergistically inhibited the growth of highly malignant +SA mammary epithelial cells in culture. Therefore, the objective of the present work was to prepare and characterize lipid nanoparticles that combined simvastatin and tocotrienol rich fraction (TRF) as potential anticancer therapy. The entrapment of simvastatin in the oily nanocompartments, which were formed by TRF inclusion into the solid matrix of the nanoparticles, was verified by its high entrapment efficiency and the absence of endothermic or crystalline peaks when blends were analyzed by DSC and PXRD, respectively. The release of simvastatin from the nanoparticles in sink conditions was characterized by an initial burst release of approximately 20% in 10h followed by a plateau. No significant change in particle size (approximately 100 nm) was observed after storage for six months. The anticancer activity of the nanoparticles was verified in vitro by observing their antiproliferative effects on malignant +SA mammary epithelial cells. The IC(50) of the reference alpha-tocopherol nanoparticles was 17.7 microM whereas the IC(50) of the simvastatin/TRF nanoparticles was 0.52 microM, which confirmed the potency of the combined treatment and its potential in cancer therapy.
A γ-tocopherol-rich mixture of tocopherols inhibits chemically induced lung tumorigenesis in A/J mice and xenograft tumor growth
Gang Lu1, Hang Xiao, Guang-Xun Li1, Sonia C.Picinich, Yu-Kuo Chen1, Anna Liu, Mao-Jung Lee, Shea Loy and Chung S.Yang
Carcinogenesis. 2010 Apr;31(4):687-94.
The present study investigated the effects of a preparation of a g-tocopherol-rich mixture of tocopherols (g-TmT) on chemically induced lung tumorigenesis in female A/J mice and the growth of H1299 human lung cancer cell xenograft tumors. In the A/J mouse model, the lung tumors were induced by either 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone (NNK; intraperitoneal injections with 100 and 75 mg/kg on Week 1 and 2, respectively) or NNK plus benzo[a]pyrene (B[a]P) (8 weekly gavages of 2 mmole each from Week 1 to 8). The NNK plus B[a]P treatment induced 21 tumors per lung on Week 19; dietary 0.3% g-TmT treatment during the entire experimental period significantly lowered tumor multiplicity, tumor volume and tumor burden (by 30, 50 and 55%, respectively; P < 0.05). For three groups of mice treated with NNK alone, the g-TmT diet was given during the initiation stage (Week 0 to 3), post-initiation stage (Week 3 to 19) or the entire experimental period, and the tumor multiplicity was reduced by 17.8, 19.7 or 29.3%, respectively (P < 0.05). g-TmT treatment during the tumor initiation stage or throughout the entire period of the experiment also significantly reduced tumor burden (by 36 or 43%, respectively). In the xenograft tumor model of human lung cancer H1299 cells in NCr-nu/nu mice, 0.3% dietary g-TmT treatment significantly reduced tumor volume and tumor weight by 56 and 47%, respectively (P < 0.05). In both the carcinogenesis and tumor growth models, the inhibitory action of g-TmT was associated with enhanced apoptosis and lowered levels of 8-hydroxydeoxyguanine, g-H2AX and nitrotyrosine in the tumors of the g-TmT-treated mice. In cell culture, the growth of H1299 cells was inhibited by tocopherols with their effectiveness following the order of d-T > g-TmT > g-T, whereas a-T was not effective. These results demonstrate the inhibitory effect of g-TmT against lung tumorigenesis and the growth of xenograft tumors of human lung cancer cells. The inhibitory activity may be due mainly to the actions of d-T and g-T.
In vivo evidence of gamma-tocotrienol as a chemosensitizer in the treatment of hormone-refractory prostate cancer
Yap WN, Zaiden N, Luk SY, Lee DT, Ling MT, Wong YC, Yap YL.
Pharmacology. 2010;85(4):248-58. Epub 2010 Apr 7.
Gamma-Tocotrienol (gammaT3) is known to selectively kill prostate cancer (PCa) cells and to sensitize the cells to docetaxel (DTX)-induced apoptosis. In the present study, the pharmacokinetics of gammaT3 and the in vivo cytotoxic response of androgen-independent prostate cancer (AIPCa) tumor following gammaT3 treatment were investigated. Here, we investigated these antitumor effects for PCa tumors in vivo. The pharmacokinetic and tissue distribution of gammaT3 after exogenous gammaT3 supplementation were examined. Meanwhile, the response of the tumor to gammaT3 alone or in combination with DTX were studied by real-time in vivo bioluminescent imaging and by examination of biomarkers associated with cell proliferation and apoptosis. After intraperitoneal injection, gammaT3 rapidly disappeared from the serum and was selectively deposited in the AIPCa tumor cells. Administration of gammaT3 alone for 2 weeks resulted in a significant shrinkage of the AIPCa tumors. Meanwhile, further inhibition of the AIPCa tumor growth was achieved by combined treatment of gammaT3 and DTX (p < 0.002). The in vivo cytotoxic antitumor effects induced by gammaT3 seem to be associated with a decrease in expression of cell proliferation markers (proliferating cell nuclear antigen, Ki-67 and Id1) and an increase in the rate of cancer cell apoptosis [cleaved caspase 3 and poly(ADP-ribose) polymerase]. Additionally, the combined agents may be more effective at suppressing the invasiveness of AIPCa. Overall, our results indicate that gammaT3, either alone or in combination with DTX, may provide a treatment strategy that can improve therapeutic efficacy against AIPCa while reducing the toxicity often seen in patients treated with DTX.
Enhanced antiproliferative and apoptotic response to combined treatment of gamma-tocotrienol with erlotinib or gefitinib in mammary tumor cells
Bachawal SV, Wali VB, Sylvester PW.
BMC Cancer. 2010 Mar 8;10:84.
Background: Aberrant ErbB receptor signaling is associated with various types of malignancies. gamma-Tocotrienol is a member of the vitamin E family of compounds that displays potent anticancer activity that is associated with suppression in ErbB receptor phosphorylation and mitogenic signaling. Erlotinib and gefitinib are tyrosine kinase inhibitors that block ErbB1 receptor activation, whereas trastuzumab is a monoclonal antibody that has been designed to specifically inhibit ErbB2 receptor activation. However, the clinical effectiveness of these agents have been disappointing because of cooperation between different ErbB family members that can rescue cancer cells from agents directed against a single ErbB receptor subtype. It was hypothesized that targeting multiple ErbB receptor subtypes with combined treatment of gamma-tocotrienol and ErbB receptor inhibitors would provide greater anticancer effects than monotherapy targeting only a single ErbB receptor subtype.
Methods: Highly malignant mouse +SA mammary epithelial cells were maintained in culture on serum-free defined media containing 10 ng/ml EGF as a mitogen. Cell viability wase determined by MTT assay, whereas Western blot and immunofluorescent staining was used to determine treatmenteffects on ErbB receptor subtype level and activation. Treatment-induced apoptosis was determined using annexin V staining and Western blot analysis of cleaved caspase-3 and PARP levels.
Results: Treatment with 3.5 microM gamma-tocotrienol, 0.5 microM erlotinib or 1.0 microM gefitinib alone, significantly inhibited +SA tumor cell growth. Combined treatment with subeffective doses of erlotinib (0.25 microM) or gefitinib (0.5 microM) with subeffective doses of gamma-tocotrienol(0.5-3.0 microM) significantly inhibited the growth and induced apoptosis in a dose-responsive manner. Trastuzumab treatment alone or in combination had no effect on +SA cell growth and viability. Combined treatment of gamma-tocotrienol with erlotinib or gefitinib also cause a large decrease in ErbB3, ErbB4, and to a lesser extent ErbB2 receptor levels, and EGF-dependent ErbB2-4 tyrosine phosphorylation (activation), but had no effect on ErbB1 receptor levels or activation.
Conclusion: Combination treatment of gamma-tocotrienol with specific ErbB receptor inhibitors is more effective in reducing mammary tumor cell growth and viability than high dose monotherapy, suggesting that targeting multiple ErbB receptors with combination therapy may significantly improve the therapeutic response in breast cancer patients.
Inhibitory effects of gamma-tocotrienol on invasion and metastasis of human gastric adenocarcinoma SGC-7901 cells
Liu HK, Wang Q, Li Y, Sun WG, Liu JR, Yang YM, Xu WL, Sun XR, Chen BQ.
J Nutr Biochem. 2010 Mar;21(3):206-13. Epub 2009 Feb 5.
Natural vitamin E is a mixture of two classes of compounds, tocopherols and tocotrienols. Recent research has revealed that tocotrienols, especially gamma-tocotrienol, exhibit not only the same antioxidant ability as tocopherols, but also remarkable anticancer capacity in cancer cell lines. In this study, the invasion and metastatic capacities of gastric adenocarcinoma SGC-7901 cells and the correlation with antimetastasis mechanisms induced by gamma-tocotrienol were explored. The results showed the inhibitory effects of gamma-tocotrienol at doses of 15, 30, 45 and 60 mumol/L for 48 h on cell migration and cell matrigel invasion; activities of matrix metalloproteinase (MMPs) increased in SGC-7901 cells when compared to the control group (P<.05 or P<.01). An increasing trend in the chemotactic responses to fibronectin (FN) in SGC-7901 cells was found in the gamma-tocotrienol treatments. SGC-7901 cell attachment decreased in the gamma-tocotrienol-treated groups in comparison with the control group (P<.01). The mRNA expressions of MMP-2 and MMP-9 showed that gamma-tocotrienol significantly reduced the matrigel invasion capability through down-regulation of the mRNA expressions of MMP-2 and MMP-9 (P<.01), and up-regulation of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 in SGC-7901 cells by treatment with gamma-tocotrienol for 48 h (P<.05). gamma-Tocotrienol also significantly increased the mRNA expression of nm23-H1 in SGC-7901 cells (P<.01). These findings suggest a potential mechanism of gamma-tocotrienol-mediated antitumor metastasis activity and indicate the role of vitamin E as potential chemopreventative agents against gastric cancer.
Nanomolar vitamin E alpha-tocotrienol inhibits glutamate-induced activation of phospholipase A2 and causes neuroprotection
Khanna S, Parinandi NL, Kotha SR, Roy S, Rink C, Bibus D, Sen CK.
J Neurochem. 2010 Mar;112(5):1249-60. Epub 2009 Dec 17.
Our previous works have elucidated that the 12-lipoxygenase pathway is directly implicated in glutamate-induced neural cell death, and that such that toxicity is prevented by nM concentrations of the natural vitamin E alpha-tocotrienol (TCT). In the current study we tested the hypothesis that phospholipase A(2) (PLA(2)) activity is sensitive to glutamate and mobilizes arachidonic acid (AA), a substrate for 12-lipoxygenase. Furthermore, we examined whether TCT regulates glutamate-inducible PLA(2) activity in neural cells. Glutamate challenge induced the release of [(3)H]AA from HT4 neural cells. Such response was attenuated by calcium chelators (EGTA and BAPTA), cytosolic PLA(2) (cPLA(2))-specific inhibitor (AACOCF(3)) as well as TCT at 250 nM. Glutamate also caused the elevation of free polyunsaturated fatty acid (AA and docosahexaenoic acid) levels and disappearance of phospholipid-esterified AA in neural cells. Furthermore, glutamate induced a time-dependent translocation and enhanced serine phosphorylation of cPLA(2) in the cells. These effects of glutamate on fatty acid levels and on cPLA(2) were significantly attenuated by nM TCT. The observations that AACOCF(3), transient knock-down of cPLA(2) as well as TCT significantly protected against the glutamate-induced death of neural cells implicate cPLA(2) as a TCT-sensitive mediator of glutamate induced neural cell death. This work presents first evidence recognizing glutamate-induced changes in cPLA(2) as a novel mechanism responsible for neuroprotection observed in response to nanomolar concentrations of TCT.