Tsuyoshi Tsuzuki

Anti-angiogenic activity of tocotrienol.

The anti-angiogenic property of vitamin E compounds, with particular emphasis on tocotrienol, has been investigated in vitro. Tocotrienol, but not tocopherol, inhibited both the proliferation and tube formation of bovine aortic endothelial cells, with δ-tocotrienol appearing the highest activity. Also, δ-tocotrienol reduced the vascular endothelial growth factor-stimulated tube formation by human umbilical vein endothelial cells. Our findings suggest that tocotrienol has potential use as a therapeutic dietary supplement for minimizing tumor angiogenesis.

Tumor angiogenesis suppression by α-eleostearic acid, a linolenic acid isomer with a conjugated triene system, via peroxisome proliferator-activated receptor γ

We have shown previously that alpha-eleostearic acid (ESA), a linolenic acid isomer with a conjugated triene system, suppresses tumor growth in vivo. In our earlier study, blood vessels were observed at the tumor surface in control mice, whereas in ESA-treated mice no such vessels were observed and the inner part of the tumor was discolored. These observations suggested that ESA might suppress cancer cell growth through malnutrition via a suppressive effect on tumor angiogenesis. In the current study, the antiangiogenic effects of ESA were investigated in vivo and in vitro. Tumor cell-induced vessel formation was clearly suppressed in mice orally administered ESA at doses of 50 and 100 mg/kg/day in a dose-dependent manner. ESA also inhibited the formation of capillary-like networks by human umbilical vein endothelial cells (HUVEC) and moderately inhibited HUVEC proliferation and migration in a dose-dependent manner. The mechanism by which ESA inhibited angiogenesis was through suppression of the expression of vascular endothelial growth factor receptors 1 and 2, activation of peroxisome proliferator-activated receptor gamma (PPARgamma) and induction of apoptosis in HUVEC. We thus demonstrated that, like troglitazone, ESA is a PPARgamma ligand and that it activates PPARgamma, induces apoptosis in HUVEC and inhibits angiogenesis. Our findings suggest that ESA has potential use as a therapeutic dietary supplement and medicine for minimizing tumor angiogenesis.

Tumor anti-angiogenic effect and mechanism of action of δ-tocotrienol

Anti-angiogenic therapy mediated by drugs and food components is an established strategy for cancer prevention. Our previous cell-culture studies identified a food-derived anti-angiogenic compound, tocotrienol (T3, an unsaturated vitamin E), as a potential angiogenic inhibitor. Among T3 isomers, delta-T3 is considered as the most potent compound. The purpose of this study was therefore to evaluate the inhibitory effect of delta-T3 on tumor angiogenesis. As growth factors (e.g., vascular endothelial growth factor and fibroblast growth factor) play critical roles in tumor angiogenesis, a conditioned medium rich in these growth factors from human colorectal adenocarcinoma cells (DLD-1-CM) was used as an angiogenic stimulus. Delta-T3 (2.5-5 microM) significantly suppressed DLD-1-CM-induced tube formation, migration, and adhesion on human umbilical vein endothelial cells. These effects were partly associated with reactive oxygen species generation by delta-T3. Western blot analysis revealed that the anti-angiogenic effect of delta-T3 is attributable to regulation of growth factor-dependent phosphatidylinositol-3 kinase (PI3K)/phosphoinositide-dependent protein kinase (PDK)/Akt signaling as well as to induction stress response in endothelial cells. Moreover, we conducted an in vivo mouse Matrigel plug angiogenesis assay, and found that delta-T3 (10-20 microg) exhibits dose-dependent inhibition of DLD-1-induced vessel formation. These results suggest that T3 has potential use as a therapeutic dietary supplement for minimizing tumor angiogenesis.

Oxidation rate of conjugated linoleic acid and conjugated linolenic acid is slowed by triacylglycerol esterification and α-tocopherol

We have recently shown that α-eleostearic acid (α-ESA), a conjugated linolenic acid, has a stronger antitumor effect than conjugated linoleic acid (CLA), both in vitro and in vivo. In this study, the oxidative stability of α-ESA was examined compared with linoleic acid (LA), α-linolenic acid (LnA), and CLA. Thin layers of the FA (LA, 9Z, 11E-CLA, 10E,12Z-CLA, LnA, and α-ESA) were auto-oxidized at 37°C, and the FA remaining, the absorbed oxygen volume, the lipid hydroperoxide content, and the TBARS content were determined. The oxidation rate of α-ESA was faster than that of the unconjugated FA and CLA (9Z,11E-CLA and 10E,12Z-CLA). However, the lipid hydroperoxide and TBARS contents following α-ESA oxidation were low, suggesting production of only small amounts of rapid-reacting secondary oxidation products. Furthermore, the oxidative stability of conjugated FA (CLA and CLnA) in which the carboxylic acid group was esterified with triacylglycerol was greater than that of the FFA. Addition of an antioxidant (α-tocopherol) also increased the stability of the conjugated FA to a level similar to that of the unconjugated FA.

Anti-angiogenic potential of tocotrienol in vitro

Modulation of angiogenesis is now a recognized strategy for the prevention of various angiogenesis-mediated disorders. We investigated, using well-characterizedin 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.

Efflux of Sphingoid Bases by P-Glycoprotein in Human Intestinal Caco-2 Cells

The aim of this study was to determine whether sphingoid bases that originated from various dietary sources, such as mammals, plants, and fungi, are substrates for P-glycoprotein in differentiated Caco-2 cells, which are used as a model of intestinal epithelial cells. In Caco-2 cells, the uptake of sphingosine, the most common sphingoid base found in mammals, was significantly higher at physiological temperatures than those of cis/trans-8-sphingenine, trans-4, cis/trans-8-sphingadienine, 9-methyl-trans-4, trans-8-sphingadienine, or sphinganine. Verapamil, a potent P-glycoprotein inhibitor, increased the cellular accumulation of sphingoid bases, except for sphingosine, in a dose-dependent manner. Incubation with 1 μM digoxin for 48 h caused up-regulation of murtidrug-resistance (MDR)1 mRNA and decreased the accumulation of sphingoid bases in Caco-2 cells, except for sphingosine. Thus P-glycoprotein probably contributes to the selective absorption of sphingosine from dietary sphingolipids in the digestive tract.

Distribution of Tocotrienols in Rats Fed a Rice Bran Tocotrienol Concentrate

To examine the distribution of rice bran tocotrienol (T3), we gave rice bran T3 to rats after considering an acceptable daily intake of vitamin E for humans. Male SD rats (5 weeks of age) were fed for 3 weeks on a commercial diet containing 6.4 mg of vitamin E per 100 g wt and additively received vitamin E or the vehicle (vitamin E-free corn oil) by oral intubation. The animals were randomly divided into 4 groups depending on the type of test diet: control (vehicle), non-T3 (no T3 + 4.3 mg of tocopherol (TOC)/kg body weight (b.w.)/day), low-T3 (0.8 mg T3 + 3.5 mg TOC/kg b.w./day), and high-T3 (3.2 mg T3 + 1.1 mg TOC/kg b.w./day). The control rats and rats in the non-T3, low-T3, and high-T3 groups took 4.3 and 8.6 mg of vitamin E/kg b.w./day, respectively. Rice bran γ-T3 was significantly distributed to the adipose tissue and increased from 1.1 to 10.2 nmol/g of adipose tissue according to the rice bran T3 intake.

Antiangiogenic potency of vitamin E

Abstract: We investigated the antiangiogenic property and mechanism of vitamin E compounds, with particular emphasis on tocotrienol (T3), a natural analogue of tocopherol (Toc). T3 inhibited both the proliferation and tube formation of bovine aortic endothelial cells, with δ‐T3 appearing to have the highest activity. δ‐T3 also reduced the vascular endothelial growth factor (VEGF)‐stimulated tube formation by human umbilical vein endothelial cells. Moreover, δ‐T3 inhibited the new blood vessel formation on the growing chick embryo chorioallantoic membrane (assay for in vivo angiogenesis). Orally administered T3 suppressed the tumor cell‐induced angiogenesis in the mouse dorsal air sac assay. In contrast with T3, Toc showed very weak inhibition. Based on DNA microarray analysis, antiangiogenic effect of T3 was attributable in part to regulation of intracellular VEGF signaling (phospholipase C‐γ and protein kinase C). Our findings suggest that T3 has potential as a therapeutic dietary supplement for preventing angiogenic disorders.

Intake of conjugated eicosapentaenoic acid suppresses lipid accumulation in liver and epididymal adipose tissue in rats.

It has been reported that consumption of CLA and EPA alters lipid metabolism. CLA contains conjugated double bonds, and EPA is an n−3 PUFA. Based on the possibility that a molecule with both of these structures might have interesting physiological effects, we prepared conjugated FA from EPA by alkaline isomerization and examined the effects of the conjugated EPA (CEPA) on lipid metabolism in rats. Rats were fed by oral gavage every day for 4 wk with 200 mg of FA including linoleic acid, EPA, CLA, or CEPA. Compared with other groups, rats fed CEPA showed a significant weight loss in epididymal adipose tissue and significant decreases in the levels of liver TAG and total cholesterol (TC), indicating reduced accumulation of lipid in the liver and adipose tissue. The plasma levels of TAG, TC, FFA, and tumor necrosis factor-α in rats fed CEPA were reduced, as was the activity of the FA synthesis system in the liver, whereas the FA-β-oxidation system was activated by CEPA. These results suggest that intake of CEPA suppresses lipid accumulation in the liver and epididymal adipose tissue while increasing lipid catabolism in rats.

Suppression of γ-tocotrienol on UVB induced inflammation in HaCaT keratinocytes and HR-1 hairless mice via inflammatory mediators multiple signaling.

Tocopherol (Toc) such as alpha-Toc has been expected to act as photochemopreventive agent of skin, but the effect of the other vitamin E forms [tocotrienols (T3)] has not been fully understood. We evaluated the anti-inflammatory effect of T3 on UVB-induced inflammatory reaction using immortalized human keratinocytes and hairless mice. gamma-T3 suppressed UVB-induced PGE(2) production while similar alpha-Toc doses had no effect. The anti-inflammatory actions of gamma-T3 were explained by its ability to reduce UVB-induced inflammatory gene and protein expression [cyclooxgenase-2 (COX-2), interleukin (IL)-1beta, IL-6, and monocyte chemotactic protein-1]. Western blot analysis revealed gamma-T3 inhibited p38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase/stress-activated protein kinase activation. In HR-1 hairless mice, oral T3 suppressed UVB-induced changes in skin thickness, COX-2 protein expression, and hyperplasia, but alpha-Toc did not. These results suggest T3 has potential use to protect against UVB-induced skin inflammation.