Tocotrienol (T3), unsaturated vitamin E, has recently gained considerable attention as a potent antiangiogenic agent minimizing tumor growth, the exact intracellular mechanisms of which remain poorly understood. Because hypoxia-inducible factor-1alpha (HIF-1alpha), its downstream target vascular endothelial growth factor (VEGF), and other angiogenic factors such as interleukin-8 (IL-8) and cyclooxygenase 2 (COX-2) play critical roles in neovascularization, we tested the hypothesis that the inhibitory effect of T3 on tumor angiogenesis is via regulation of these angiogenic factors. We used 2 cancer cell lines, human colorectal adenocarcinoma cells (DLD-1) and human hepatoma cells (HepG2). T3 isomers (2 micromol/L) inhibited hypoxia-induced VEGF secretion from DLD-1, with delta-T3 showing potent inhibition. Delta-T3 suppressed hypoxia-induced VEGF and IL-8 expression in DLD-1 at both mRNA and protein levels, and we found the inhibitory mechanism of delta-T3 by reducing HIF-1alpha protein expression or increasing HIF-1alpha degradation. Also, delta-T3 (2 micromol/L) did not affect hypoxia-induced COX-2 mRNA expression; however, delta-T3 tended to suppress (P = 0.044) hypoxia-induced COX-2 protein expression, implying a possible post-transcriptional mechanism by delta-T3. Overall, our results confirmed that T3 has an inhibitory effect on angiogenic factor secretion from cancer cells and revealed the possible mechanisms, providing new information about the antiangiogenic effects of T3.

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Natural compounds with possible health benefits have become attractive targets for research in areas pertaining to human health. For both prevention and therapy of various human ailments, such compounds are preferred over synthetic ones due to their lesser toxicity. They are also easily absorbed and processed by our body. Vitamins are prominent among natural or endogenous compounds that are considered to be beneficial. The vitamin E group of compounds is among the better known of the vitamins due to their suggested health benefits including antioxidant and related protective properties. Among these, tocotrienols have gained prominence in recent years due to their potential applications and better protective effects in certain systems. These tocotrienols are vitamin E derivatives that are analogs of the more established forms of vitamin E namely tocopherols. In addition to their potent antioxidant activity, tocotrienols have other important functions, especially in maintaining a healthy cardiovascular system and a possible role in protection against cancer and other ailments.

Tocotrienol is an antioxidant which has found commercial application as a food additive and health supplement all over the world. Since there have been no reports regarding toxicological effects of long-term exposure, we performed a 52-week chronic study using Wistar Hannover rats of both sexes given the compound at doses of 0, 0.08, 0.4 or 2% in powdered basal diet. Since 6 animals in the 2% male group died of hemorrhage of several organs by week 50, the maximum dose level was changed to 1% in both sexes for the last 2 weeks. Decrease of body weight gain was observed in the 2% males from week 5 and females from week 10, this persisting to the end of the study. With the high dose, prolongation of prothrombin time and increase of serum ALT in males, and increase of serum ALP in both sexes were observed with statistical significance. In male and female rats receiving 0.4% or less, there were no toxicological changes in any of the parameters examined. At necropsy, multiple cyst-like nodules on the liver surface were macroscopically pronounced in both sexes receiving 2%. On histopathological examination, hepatocellular nodules were evident with distortion of hepatic cords and compression of the surrounding tissue, almost all including areas of spongiosis hepatis. The constituent hepatocytes were immunohistochemically stained with proliferation cell nuclear antigen at high rates. Nevertheless, they did not exhibit overt atypia and the basic lobular architecture remained intact. Additionally, they were consistently negative for glutathione S-transferase placental form (GST-P). Accordingly, we propose the newly categorized but previously used name ‘nodular hepatocellular hyperplasia’, which may not necessarily have a neoplastic or regenerative nature. However, quantitative GST-P analysis of the liver sections overall showed numbers of GST-P foci in the high dose females to be significantly elevated as compared to the control value. Based on the present data demonstrating nodular liver lesions only at the high dose of both sexes, we conclude that the no-observed-adverse-effect level (NOAEL) is 0.4% (303 mg/kg/day for males, and 472 mg/kg/day for females).

Current observations in the literature suggest that vitamin E may be a suitable candidate for the adjuvant treatment of cancer. Even though historically most research focused on alpha-tocopherol, more recent evidence suggests that the other isomers of vitamin E (beta-, gamma- and delta-tocopherols and alpha-, beta-, gamma- and delta-tocotrienols) differ in their proapoptotic potencies. The main focus of this communication is the current understanding of the molecular mechanisms regulated by vitamin E isomers and their analogs during the induction of apoptosis. This review highlights that the mitochondria are the major target for the induction of apoptosis by vitamin E isomers and analogs and that the various signaling pathways regulated by these agents are likely to contribute towards maximizing the intrinsic pathway of apoptosis triggered initially by the mitochondria. Overall, the presentation of recent studies from the literature in this communication allows the drawing of the following important conclusions: (i) no direct link exists between the antioxidant activity of each isomer/derivative and proapoptotic potency, (ii) tocotrienols are more effective proapoptotic agents than tocopherols, (iii) synthetic modifications of the naturally occurring compounds may improve their apoptotic potency and (iv) vitamin E isomers and derivatives regulate caspase-independent pathways of apoptosis. The latter combined with the evidence presented in this review regarding the additive or synergistic anticarcinogenic effects obtained when vitamin E analogs are used in combination with other cancer chemotherapeutic agents, supports further research to design the most promising vitamin E derivatives and clinically test them in adjuvant chemotherapeutic treatments.

Tocotrienols have been shown to inhibit proliferation and induce apoptosis in cancer cells. However, the molecular mechanisms involved in tocotrienol-induced apoptosis are still unclear. In the present study, gamma-tocotrienol induced apoptosis in human gastric adenocarcinoma SGC-7901 cell line through down regulation of the extracellular signal-regulated kinase (ERK) signalling pathway. Furthermore, gamma-tocotrienol-induced apoptosis was accompanied by down regulation of Bcl-2, up regulation of Bax, activation of caspase-3, and subsequent poly (ADP-ribose) polymerase cleavage. These results indicated that up or down regulation of Bcl-2 family proteins play a major role in the initiation of gamma-tocotrienol-induced apoptosis as an activator of caspase-3. Gamma-tocotrienol also down regulated the activation of the Raf-ERK signalling pathway, and down regulated c-Myc by decreasing the expressions of Raf-1 and p-ERK1/2 proteins. The results suggest that key regulators in tocotrienol-induced apoptosis may be Bcl-2 families and caspase-3 in SGC-7901 cells through down regulation of the Raf-ERK signalling pathway.

Vitamin E is a generic term that refers to a family of compounds that is further divided into two subgroups called tocopherols and tocotrienols. All natural forms of tocopherols and tocotrienols are potent antioxidants that regulate peroxidation reactions and controls free radical production within the body. However, it is now firmly established that many of the biological actions mediated by individual vitamin E isoforms are not dependent on their antioxidant activity. Furthermore, synthetic ether derivatives of vitamin E that no longer possess antioxidant activity also display a wide range of biological activities. One of the most intriguing therapeutic applications for natural vitamin E and vitamin E derivatives currently being investigated is their use as anticancer agents. Specific forms of vitamin E display potent apoptotic activity against a wide range of cancer cell types, while having little or no effect on normal cell function or viability. Experimental studies have also determined that the intracellular mechanisms mediating the apoptotic effects of specific vitamin E compounds display great diversity in different types of caner cells and has been found to restore multidrug resistant tumor cells sensitivity to chemotherapeutic agents. These findings strongly suggest that some natural and synthetic analogues of vitamin E can be used effectively as anticancer therapy either alone or in combination to enhance the therapeutic efficacy and reduce toxicity of other anticancer agents.

Antiangiogenic therapy using drugs and food components is a recognized strategy for the prevention of various angiogenesis-mediated disorders such as tumor growth, diabetic retinopathy, and rheumatoid arthritis. Our preliminary cell culture studies, using both bovine aortic endothelial cells and human umbilical vein endothelial cells (HUVEC) on screening for food-derived antiangiogenic compounds, showed tocotrienol (T3), an unsaturatedversion of vitamin E, to be a potential angiogenic inhibitor. We therefore investigaged the in vivo antiangiogenic properties of T3 using 2 well-characterized angiogenic models [mouse dorsal air sac (DAS) assay and the chick embryo chorioallantoic membrane (CAM) assay]. In the DAS assay, the increased neovascularization (angiogenesis index, 4.8 +/- 0.6) in tumor cell-implanted mice was suppressed (angiogenesis index, 2.7 +/- 0.6) by dietary supplementation of 10 mg T3-rich oil/d (equivalent to 4.4 mg T3/d). In the CAM assay, T3 (500-1000 microg/egg) inhibited new blood vessel formation on the growing CAM and increased the frequency of avascular zone (36-50%). To evaluate the antiangiogenic mechanism, we conducted cell-culture studies and found that T3 significantly reduced fibroblast growth factor -induced proliferation, migration, and tube formation inHUVEC (P < 0.05), with delta-T3 having the highest activity. Western blot analysis revealed that delta-T3 suppressed the phosphorylation of phosophoinositide-dependent protein kinase (PDK) and Akt, and increased the phosphorylation of apoptosis signal-regulating kinase and p38 infibroblast growth factor-treated HUVEC, indicating that the antiangiogenic effects of T3 are associated with changes in growth factor-dependent phosphatidylinositol-3 kinase /PDK/Akt signaling as well as induction of apoptosis in endothelial cells. Our findings suggest that T3 has potential as a therapeutic dietary supplement for preventing angiogenic disorders, and therefore future clinical study will be required to evaluate the efficacy and safety of T3.

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3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is the rate-limiting enzyme in the mevalonate pathway that provides essential intermediates for the membrane anchorage and biologic functions of growth-related proteins. Contrary to preclinical studies showing the growth-suppressive activity of statins, competitive inhibitors of HMG CoA reductase, clinical application of statins in cancer is precluded by their lack of activity at levels prescribed for the prevention of cardiovascular disease and by their dose-limiting toxicities at high doses. The dysregulated and elevated HMG CoA reductase activity in tumors retains sensitivity to the isoprenoid-mediated posttranscriptional down-regulation, an action that complements the statin-mediated inhibition and may lead to synergistic impact of blends of isoprenoids and lovastatin on tumor HMG CoA reductase activity and consequently tumor growth. d-gamma- and d-delta-tocotrienols, vitamin E isomers containing an isoprenoid moiety, and lovastatin-induced concentration-dependent inhibition of the 48-hr proliferation of murine B16 melanoma cells with IC50 values of 20 +/- 3, 14 +/- 3, and 1.5 +/- 0.4 microM respectively. A blend of lovastatin (1 microM) and d-gamma-tocotrienol (5 microM) totally blocked cell growth, an impact far exceeding the sum of inhibitions induced by lovastatin (12%) and d-gamma-tocotrienol (8%) individually. Synergistic impact of these two agents was also shown in human DU145 prostate carcinoma and human A549 lung carcinoma cells. C57BL6 mice were fed diets supplemented with 12.5 mg lovastatin/kg body weight, 62.5 mg d-delta-tocotrienol/kg body weight, or a blend of both agents for 22 days following B16 cell implantation; only the latter had significantly lower tumor weight than those with no supplementation. Co-administration of isoprenoids that posttranscriptionally down-regulate tumor reductase may lower the effective dose of statins and offer a novel approach to cancer chemo-prevention and/or therapy.

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Unlike the tocopherols, the tocotrienols, also members of the vitamin E family, have an unsaturated isoprenoid side chain. In contrast to extensive studies on tocopherol, very little is known about tocotrienol. Because the nuclear factor-kappaB (NF-kappaB) pathway has a central role in tumorigenesis, we investigated the effect of gamma-tocotrienol on the NF-kappaB pathway. Although gamma-tocotrienol completely abolished tumor necrosis factor alpha (TNF)-induced NF-kappaB activation, a similar dose of gamma-tocopherol had no effect. Besides TNF, gamma-tocotrienol also abolished NF-kappaB activation induced by phorbol myristate acetate, okadaic acid, lipopolysaccharide, cigarette smoke, interleukin-1beta, and epidermal growth factor. Constitutive NF-kappaB activation expressed by certain tumor cells was also abrogated by gamma-tocotrienol. Reducing agent had no effect on the gamma-tocotrienol-induced down-regulation of NF-kappaB. Mevalonate reversed the NF-kappaB inhibitory effect of gamma-tocotrienol, indicating the role of hydroxymethylglutaryl-CoA reductase. Gamma-tocotrienol blocked TNF-induced phosphorylation and degradation of IkappaBalpha through the inhibition of IkappaBalpha kinase activation, thus leading to the suppression of the phosphorylation and nuclear translocation of p65. gamma-Tocotrienol also suppressed NF-kappaB-dependent reporter gene transcription induced by TNF, TNFR1, TRADD, TRAF2, TAK1, receptor-interacting protein, NIK, and IkappaBalpha kinase but not that activated by p65. Additionally, the expressions of NF-kappaB-regulated gene products associated with antiapoptosis (IAP1, IAP2, Bcl-xL, Bcl-2, cFLIP, XIAP, Bfl-1/A1, TRAF1, and Survivin), proliferation (cyclin D1, COX2, and c-Myc), invasion (MMP-9 and ICAM-1), and angiogenesis (vascular endothelial growth factor) were down-regulated by gamma-tocotrienol. This correlated with potentiation of apoptosis induced by TNF, paclitaxel, and doxorubicin. Overall, our results demonstrate that gamma-tocotrienol inhibited the NF-kappaB activation pathway, leading to down-regulation of various gene products and potentiation of apoptosis.

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