Synergistic anticancer effects of combined gamma-tocotrienol and celecoxib treatment are associated with suppression in Akt and NFkappaB signaling

Shirode AB, Sylvester PW.

Biomed Pharmacother. 2010 May;64(5):327-32. Epub 2009 Nov 14.

The selective cyclooxygenase (COX)-2 inhibitor, celecoxib, and the vitamin E isoform, gamma-tocotrienol, both display potent anticancer activity. However, high dose clinical use of selective COX-2 inhibitors has been limited by gastrointestinal and cardiovascular toxicity, whereas limited absorption and transport of gamma-tocotrienol by the body has made it difficult to obtain and sustain therapeutic levels in the blood and target tissues. Studies were conducted to characterize the synergistic anticancer antiproliferative effects of combined low dose celecoxib and gamma-tocotrienol treatment on mammary tumor cells in culture. The highly malignant mouse +SA mammary epithelial cells were maintained in culture on serum-free defined control or treatment media. Treatment effects on COX-1, COX-2, Akt, NFkappaB and prostaglandin E(2) (PGE(2)) synthesis were assessed following a 3- or 4-day culture period. Treatment with 3-4 microM gamma-tocotrienol or 7.5-10 microM celecoxib alone significantly inhibited +SA cell growth in a dose-responsive manner. However, combined treatment with subeffective doses of gamma-tocotrienol (0.25 microM) andcelecoxib (2.5 microM) resulted in a synergistic antiproliferative effect, as determined by isobologram analysis, and this growth inhibitory effect wasassociated with a reduction in PGE(2) synthesis, and decrease in COX-2, phospho-Akt (active), and phospho-NFkappaB (active) levels. These results demonstrate that the synergistic anticancer effects of combined celecoxib and gamma-tocotrienol therapy are mediated by COX-2 dependent and independent mechanisms. These findings also suggest that combination therapy with these agents may provide enhanced therapeutic response in breast cancer patients, while avoiding the toxicity associated with high-dose COX-2 inhibitor monotherapy.

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Tocopherols and tocotrienols have been originally identified as essential nutrients in mammals based on their vitamin E activity. These lipid-soluble compounds are potent antioxidants that protect polyunsaturated fatty acids from lipid peroxidation. The biosynthesis of tocopherols and tocotrienolsoccurs exclusively in photosynthetic organisms. The biosynthetic precursors and the different pathway intermediates have been identified by biochemical studies and the different vitamin E biosynthetic genes (VTE genes) have been isolated in several plants and cyanobacteria. The characterization of transgenic plants overexpressing one or multiple VTE genes combined with the study of vitamin E deficient mutants allows from now on understanding the regulation and the function of tocopherols and tocotrienols in plants.

Vitamin E is composed of closely related compounds, including tocopherols and tocotrienols. Studies of the last decade provide strong support for a specific role of alpha-tocopherol in cell signalling and the regulation of gene expression. It produces significant effects on inflammation, cell proliferation and apoptosis that are not shared by other vitamin E isomers with similar antioxidant properties. The different behaviours of vitamin E isomers might relate, at least in part, to the specific effects they exert at the plasma membrane. alpha-Tocopherol is not randomly distributed throughout the phospholipid bilayer of biological membranes, and as compared with other isomers, it shows a propensity to associate with lipid rafts. Distinct aspects of vitamin E transport and metabolism is discussed with emphasis on the interaction between alpha-tocopherol and lipid rafts and the consequences of these interactions on cell metabolism.

Vitamin E refers to a family of several compounds that possess a similar chemical structure comprising a chromanol ring with a 16-carbon side chain. The degree of saturation of the side chain, and positions and nature of methyl groups designate the compounds as tocopherols ortocotrienols. Vitamin E compounds have antioxidant properties due to a hydroxyl group on the chromanol ring. Recently, it has been suggested that vitamin E may also regulate signal transduction and gene expression. We previously reported that lifelong dietary vitamin E (alpha-tocopherol) supplementation significantly increased median lifespan in C57BL/6 mice by 15%. This lifespan extension appeared to be independent of any antioxidant effect. Employing a transcriptional approach, we suggest that this increase in lifespan may reflect an anti-cancer effect via induction of the P21 signalling pathway, since cancer is the major cause of death in small rodents. We suggest that the role of this pathway in life span extension following supplementation of vitamin E now requires further investigation.

In vitro antioxidant activity of tocopherols and tocotrienols and comparison of Vitamin E concentration and lipophilic antioxidant capacity in human plasma

Müller L, Theile K, Böhm V.

Mol Nutr Food Res. 2010 May;54(5):731-42.

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.

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.