Disruption of mitochondria during tocotrienol-induced apoptosis in MDA-MB-231 human breast cancer cells

Takahashi K, Loo G.

Biochem Pharmacol. 2004 Jan 15;67(2):315-24.

Tocotrienols, which are Vitamin E isoforms, are known to inhibit the growth of human breast cancer cells due partly to apoptosis. However, the characterization of tocotrienol-induced apoptosis is incomplete, particularly what happens during the initiation phase that precedes execution of the cells. The objective of this study was to clarify the apoptotic effects of tocotrienols, with especial emphasis in determining if the mitochondria-mediated death pathway is activated when human breast cancer cells are incubated with a specific tocotrienol isomer. During incubation with gamma-tocotrienol, MDA-MB-231 human breast cancer cells showed membrane blebbing, and apoptotic bodies were present. Upon 4′,6-diamidino-2-phenylindole staining of the cells, chromatin condensation and fragmentation were observed. Additionally, the annexin V-binding assay detected the translocation of membrane phospholipid during earlier analysis of the cells. Taken together, these results further establish that gamma-tocotrienol can induce apoptosis in human breast cancer cells. To help elucidate how gamma-tocotrienol induced the apoptosis, some important parameters related to the mitochondria-mediated death pathway were examined next. In gamma-tocotrienol-treated cells, the mitochondria were disrupted. Collapse of the mitochondrial membrane potential was detected, and cytochrome c was released later from mitochondria. However, expression of Bax and Bcl-2 (mRNA and protein) did not change. Furthermore, poly-(ADP-ribose)-polymerase cleavage was not detected, suggesting that caspases were not involved in the gamma-tocotrienol-induced apoptosis. These results imply that cytochrome c is not the critical protein released from mitochondria that triggers gamma-tocotrienol-induced apoptosis in MDA-MB-231 cells.

Anti-angiogenic potential of tocotrienol in vitro

Miyazawa T, Inokuchi H, Hirokane H, Tsuzuki T, Nakagawa K, Igarashi M.

Biochemistry (Mosc). 2004 Jan;69(1):67-9.

Modulation of angiogenesis is now a recognized strategy for the prevention of various angiogenesis-mediated disorders. We investigated, using well-characterized in vitro systems, the anti-angiogenic property of vitamin E compounds, with particular emphasis on tocotrienol, a natural analog of tocopherol. Tocotrienol, but not tocopherol, inhibited the proliferation of bovine aortic endothelial cells in dose dependent manner at half-maximal concentrations in the low micromolar range. Tocotrienol also significantly inhibited the formation of networks of elongated endothelial cells within 3D collagen gels. From these results, we suggest that tocotrienol is a potential candidate for the development of useful therapeutic agents or preventive food factors for tumor angiogenesis.

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We present here current data on the distribution and metabolism of vitamin E analogs in vivo. There are eight different naturally occurring forms of vitamin E : four tocopherols (α-,β, γ- and δ-toc) and four tocotrienols (α-,β-,γ and δ-toc). With regard to the bioavailability of vitamin E, it has been established that the affinity of various vitamin E analogs for α–tocopherol transfer protein (α-TTP), which may determine their plasma levels, is a major determinant if their biological activity however, a novel function of toc-3 has been noted as a result of its unique distribution in the skin and the adipose tissue. In addition, following the discovery that the final metabolites of Toc and Toc-3 are in the form of carboxyethyl hydroxychroman, it is now possible to examine the intermediary metabolites of vitamin E analogs. The metabolites of vitamin E is known to be involved in the actions of drug metabolic enzymes (CYP3A, CYP4F2). However, the relationship between α –TTP and the metabolic enzymes that are responsible for the regulation of vitamin E metabolism has yet to be clarified. Future research will focus on the elucidation of the vitamin E metabolic regulation system.

Tocochromanols (tocopherols and tocotrienols), collectively known as vitamin E, are essential antioxidant components of both human and animal diets. Because of their potential health benefits, there is a considerable interest in plants with increased or customized vitamin E content. Here, we have explored a new strategy to reach this goal. In plants, phenylalanine is the precursor of a myriad of secondary compounds termed phenylpropanoids. In contrast, much less carbon is incorporated into tyrosine that provides p-hydroxyphenylpyruvate and homogentisate, the aromatic precursors of vitamin E. Therefore, we intended to increase the flux of these two compounds by deriving their synthesis directly at the level of prephenate. This was achieved by the expression of the yeast (Saccharomyces cerevisiae) prephenate dehydrogenase gene in tobacco (Nicotiana tabacum) plants that already overexpress the Arabidopsis p-hydroxyphenylpyruvate dioxygenase coding sequence. A massive accumulation of tocotrienols was observed in leaves. These molecules, which were undetectable in wild-type leaves, became the major forms of vitamin E in the leaves of the transgenic lines. An increased resistance of the transgenic plants toward the herbicidal p-hydroxyphenylpyruvate dioxygenase inhibitor diketonitril was also observed. This work demonstrates that the synthesis of p-hydroxyphenylpyruvate is a limiting step for the accumulation of vitamin E in plants.
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