DNA chip analysis of comprehensive food function: Inhibition of angiogenesis and telomerase activity with unsaturated vitamin E, tocotrienol

Nakagawa K, Eitsuka T, Inokuchi H, Miyazawa T.

Biofactors. 2004;21(1-4):5-10.

Inhibition of angiogenesis and telomerase activity with vitamin E compounds, especially for tocotrienol (T3), has been investigated. Nutrigenomic tools have been used for elucidating the bioactive mechanisms of T3. In the cell culture experiments, T3 reduced the vascular endothelial growth factor (VEGF)-stimulated tube formation by human umbilical vein endothelial cells (HUVEC). Among T3 isomers, delta-T3 appeared the highest activity. The T3 inhibited the new blood vessels formation on the growing chick embryo chorioallantoic membrane (CAM assay for an in vivo model of angiogenesis). In contrast, tocopherol did not. The findings suggested that the T3 has potential use for reducing angiogenic disorder. DNA chip analysis revealed that T3 specifically down-regulates the expression of VEGF receptor (VEGFR) in endothelial cells. It is well-known that VEGF regulates angiogenesis by binding to VEGFR. Therefore, T3 could block the intracellular signaling of VEGF via down-regulation of VEGFR, which resulted in the inhibition of angiogenesis. On the other hand, DNA chip analysis also revealed that T3 down-regulates the expression of protein kinase C (PKC) in the cultured HUVEC. Since PKC is involved with the control of telomerase activity, T3 has potential to act as anti-telomerase inhibitor via PKC inhibition. In this manner, DNA chip technology provides efficient access to genetic information regarding food function and its mechanism.

Studies of the isoprenoid-mediated inhibition of mevalonate synthesis applied to cancer chemotherapy and chemoprevention

Mo H, Elson CE.

Exp Biol Med (Maywood). 2004 Jul;229(7):567-85.

Pools of farnesyl diphosphate and other phosphorylated products of the mevalonate pathway are essential to the post-translational processing and physiological function of small G proteins, nuclear lamins, and growth factor receptors. Inhibitors of enzyme activities providing those pools, namely, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase and mevalonic acid-pyrophosphate decarboxylase, and of activities requiring substrates from the pools, the prenyl protein transferases, have potential for development as novel chemotherapeutic agents. Their potentials as suggested by the clinical responses recorded in Phase I and II investigations of inhibitors of HMG CoA reductase (the statins), of mevalonic acid-pyrophosphate decarboxylase (sodium phenylacetate and sodium phenylbutyrate), and of farnesyl protein transferase (R115777, SCH66336, BMS-214662, Tipifarnib, L-778,123, and, prematurely, perillyl alcohol) are dimmed by dose-limiting toxicities. These nondiscriminant growth-suppressive agents induce G1 arrest and initiate apoptosis and differentiation, effects attributed to modulation of cell signaling pathways either by modulating gene expression, suppressing the post-translational processing of signaling proteins and growth factor receptors, or altering diacylglycerol signaling. Diverse isoprenoids and the HMG CoA reductase inhibitor, lovastatin, modulate cell growth, induce cell cycle arrest, initiate apoptosis, and suppress cellular signaling activities. Perillyl alcohol, the isoprenoid of greatest clinical interest, initially was considered to inhibit farnesyl protein transferase; follow-up studies revealed that perillyl alcohol suppresses the synthesis of small G proteins and HMG CoA reductase. In sterologenic tissues, sterol feedback control, mediated by sterol regulatory element binding proteins (SREBPs) 1a and 2, exerts the primary regulation on HMG CoA reductase activity at the transcriptional level. Secondary regulation, a nonsterol isoprenoid-mediated fine-tuning of reductase activity, occurs at the levels of reductase translation and degradation. HMG CoA reductase activity in tumors is elevated and resistant to sterol feedback regulation, possibly as a consequence of aberrant SREBP activities. Nonetheless, tumor reductase remains sensitive to isoprenoid-mediated post-transcriptional downregulation. Farnesol, an acyclic sesquiterpene, and farnesyl homologs, gamma-tocotrienol and various farnesyl derivatives, inhibit reductase synthesis and accelerate reductase degradation. Cyclic monoterpenes, d-limonene, menthol and perillyl alcohol and beta-ionone, a carotenoid fragment, lower reductase mass; perillyl alcohol and d-limonene lower reductase mass by modulating translational efficiency. The elevated reductase expression and greater demand for nonsterol products to maintain growth amplify the susceptibility of tumor reductase to isoprenoids, therein rendering tumor cells more responsive than normal cells to isoprenoid-mediated growth suppression. Blends of lovastatin, a potent nondiscriminant inhibitor of HMG CoA reductase, and gamma-tocotrienol, a potent isoprenoid shown to post-transcription-ally attenuate reductase activity with specificity for tumors, synergistically affect the growth of human DU145 and LNCaP prostate carcinoma cells and pending extensive preclinical evaluation, potentially offer a novel chemotherapeutic strategy free of the dose-limiting toxicity associated with high-dose lovastatin and other nondiscriminant mevalonate pathway inhibitors.

Induction of apoptosis by tocotrienol in rat hepatoma dRLh-84 cells

Sakai M, Okabe M, Yamasaki M, Tachibana H, Yamada K

Anticancer Res. 2004 May-Jun;24(3a):1683-8.

Our aim was to evaluate the antitumor activities of tocopherol (Toc) and tocotrienol (T3) derivatives. At first, we examined the effect of these vitamin E homologues on the proliferation of rat normal hepatocyte RLN-10 and hepatoma dRLh-84 cells and found that especially T3 inhibited cell proliferation in dRLh-84 cells. Then, we examined the effect of vitamin E homologues on apoptosis induction and found that T3 induced DNA fragmentation and stimulated a rise of caspase-3 activity. In addition, T3 stimulated a rise in caspase-8 activity, while a caspase-8 inhibitor suppressed apoptosis induction by T3. We also examined the incorporation of vitamin E homologues into dRLh-84 cells. T3 was incorporated more quickly compared to Toc. These results indicated that T3 induces apoptosis in dRLh-84 cells and that caspase-8 is involved in this apoptosis induction. The difference in terms of apoptosis induction by vitamin E homologues seems to be related to their different rates of cellular incorporation.

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Tocotrienol-rich fraction of palm oil activates p53, modulates Bax/Bcl2 ratio and induces apoptosis independent of cell cycle association

Agarwal MK, Agarwal ML, Athar M, Gupta S.

Cell Cycle. 2004 Feb;3(2):205-11.

Anti-cancer properties of palm oil have been attributed to the presence of tocotrienols and carotenoids. Studies from various laboratories have shown that tocotrienol-rich fraction (TRF) of palm oil inhibits cell growth and induces apoptosis in both preneoplastic and neoplastic cells. However, the mechanism by which TRF induces apoptosis remains largely unknown. Since several chemopreventive agents have been shown to utilize p53 pathway in negative regulation of cell growth, using human colon carcinoma RKO cells which express wild type p53, we investigated the effect of TRF on components of p53 signaling network. Treatment of cells with TRF resulted in a dose- and time- dependent inhibition of growth and colony formation. Further, TRF treatment of RKO cells resulted in the induction of WAF1/p21 which appears to be independent of cell cycle regulation and is transcriptionally upregulated in p53 dependent fashion. These results were further confirmed by using cells that express luciferase from a p53 responsive promoter where TRF treatment leads to activation of p53 reporter activity. TRF treatment also resulted in alteration in Bax/Bcl2 ratio in favor of apoptosis, which was associated with the release of cytochrome c and induction of apoptotic protease-activating factor-1. This altered expression of Bcl2 family members triggered the activation of initiator caspase-9 followed by activation of effector caspase-3. These signaling cascades lead to condensed chromatin, DNA fragmentation and shrinkage of cell membrane resulting into apoptosis. Our data suggest that TRF-induced apoptosis in colon carcinoma cells is mediated by p53 signaling network which appears to be independent of cell cycle association.

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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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Suppression of 7,12-dimethylbenz[alpha]anthracene-induced carcinogenesis and hypercholesterolaemia in rats by tocotrienol-rich fraction isolated from rice bran oil

Iqbal J, Minhajuddin M, Beg ZH.

Eur J Cancer Prev. 2003 Dec;12(6):447-53.

The anti-tumour and anti-cholesterol impacts of tocotrienol-rich fraction (TRF) were investigated in rats treated with the chemical carcinogen 7,12-dimethylbenz [alpha]anthracene (DMBA), which is known to induce mammary carcinogenesis and hypercholesterolaemia. DMBA administration to rats was associated with the appearance of multiple tumours on mammary glands after 6 months. Alkaline phosphatase (ALP) and glutathione-S-transferase (GST) are used as marker enzymes to monitor the severity of carcinogenesis. Although no tumours were visible on livers, hepatic ALP and GST activities of DMBA-treated rats were profoundly elevated in comparison to enzyme activities of normal control rats. Feeding of TRF (10 mg/kg body weight/day) for 6 months, isolated from rice bran oil (RBO), to DMBA-administered rats, reduced the severity and extent of neoplastic transformation in the mammary glands. Similarly, plasma and mammary ALP activities increased during carcinogenesis (95% and 43%, respectively), were significantly decreased in TRF-treated rats, whereas TRF mediated a further increase of 51% in hepatic ALP activity. TRF treatment to rats maintained low levels of GST activities in liver ( approximately 32%) and mammary glands ( approximately 21%), which is consistent with anti-carcinogenic properties of TRF. Administration of DMBA also caused a significant increase of 30% in plasma total cholesterol and 111% in LDL-cholesterol levels compared with normal control levels. Feeding of TRF to rats caused a significant decline of 30% in total cholesterol and 67% in LDL-cholesterol levels compared with the DMBA-administered rats. The experimental hypercholesterolaemia caused a significant increase in enzymatic activity (23%) and protein mass (28%) of hepatic 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductase. Consistent with TRF-mediated reduction in plasma lipid levels, enzymatic activity and protein mass of HMG-CoA reductase was significantly reduced. These results indicate that TRF has potent anti-cancer and anti-cholesterol effects in rats.

Anti-angiogenic activity of tocotrienol

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

Biosci Biotechnol Biochem. 2003 Jul;67(7):1623-7.

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 delta-tocotrienol appearing the highest activity. Also, delta-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.

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Vitamin E analogues as inducers of apoptosis: Structure-function relation

Birringer M, EyTina JH, Salvatore BA, Neuzil J.

Br J Cancer. 2003 Jun 16;88(12):1948-55.

Recent results show that alpha-tocopheryl succinate (alpha-TOS) is a proapoptotic agent with antineoplastic activity. As modifications of the vitamin E (VE) molecule may affect its apoptogenic activity, we tested a number of newly synthesised VE analogues using malignant cell lines. Analogues of alpha-TOS with lower number of methyl substitutions on the aromatic ring were less active than alpha-TOS. Replacement of the succinyl group with a maleyl group greatly enhanced the activity, while it was lower for the glutaryl esters. Methylation of the free succinyl carboxyl group on alpha-TOS and delta-TOS completely prevented the apoptogenic activity of the parent compounds. Both Trolox and its succinylated derivative were inactive. alpha-tocotrienol (alpha-T3 H) failed to induce apoptosis, while gamma-T3 H was apoptogenic, and more so when succinylated. Shortening the aliphatic side chain of gamma-T3 by one isoprenyl unit increased its activity. Neither phytyl nor oleyl succinate caused apoptosis. These findings show that modifications of different functional moieties of the VE molecule can enhance apoptogenic activity. It is hoped that these observations will lead to the synthesis of analogues with even higher apoptogenic and, consequently, antineoplastic efficacy.

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Palm oil alleviates 12-O-tetradecanoyl-phorbol-13-acetate-induced tumor promotion response in murine skin

Kausar H, Bhasin G, Zargar MA, Athar M.

Cancer Lett. 2003 Mar 31;192(2):151-60.

Palm oil is a rich source of vitamin E, carotenoids, tocotrienols and tocopherols which are natural antioxidants and act as scavengers of oxygen free radicals. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA) is a known oxidant that promotes tumorigenesis in mouse skin through the elaboration of oxidative stress. In this study we therefore assessed the anti-tumor promoting potential of palm oil against TPA-mediated skin tumorigenesis in 7,12-dimethylbenz[a]anthracene-initiated Swiss albino mice. Topical application of palm oil 1 h prior to application of TPA resulted in a significant protection against skin tumor promotion. The animals pre-treated with palm oil showed a decrease in both tumor incidence and tumor yield as compared to the TPA (alone)-treated group. Palm oil application also reduced the development of malignant tumors. Since TPA-induced epidermal ornithine decarboxylase (ODC) activity and [(3)H]thymidine incorporation are conventionally used markers of skin tumor promotion, we also assessed the effect of pre-application of palm oil on these parameters, and it was observed that the application of palm oil prior to the application of TPA alleviated both these TPA-induced markers of tumor promotion. The effect of pre-application of palm oil on TPA-mediated depletion in the non-enzymatic and enzymatic molecules was also assessed and it was observed that palm oil application prior to TPA application resulted in the recovery of TPA-mediated depletion in the levels of these molecules viz. glutathione, glutathione peroxidase, glutathione reductase, glutathione-S-transferase and catalase. Similarly, palm oil also exhibited a protective effect against Fe(2+)-ascorbate-induced lipid peroxidation in the epidermal microsomes. The results of the present study thus suggest that palm oil possesses anti-skin tumor promoting effects, and that the mechanism of such effects may involve the inhibition of tumor promoter-induced epidermal ODC activity, [(3)H]thymidine incorporation and cutaneous oxidative stress.

Oxidative damage to mitochondria in normal and cancer tissues, and its modulation

Kamat JP, Devasagayam TP.

Toxicology. 2000 Nov 30;155(1-3):73-82.

Cellular damage induced by reactive oxygen species (ROS) in normal tissues has been implicated in the etiology of several human ailments. Among the subcellular organelles, damage to mitochondria is considered crucial and can lead to cytotoxicity and cell death. However, the same damage, if it is selectively induced in cancer tissues can lead to its cure. Hence analyzing the mechanisms of such damage and its modulation may result in better prevention or cure. Using mitochondria derived from rat brain/liver as well as sarcoma 180 ascites cells, we have examined the mechanisms of damage to lipid, as assessed by different products of lipid peroxidation and to proteins, as determined by loss of enzyme activity and protein oxidation. Mechanisms involved, in terms of scavenging of ROS have been determined using pulse radiolysis for hydroxyl radical and histidine destruction assay for singlet oxygen. Various ROS were generated using gamma-radiation, photosensitization etc. under different conditions. Some novel porphyrins, with potential uses in photodynamic therapy also were used as photosensitizers. Our study shows that ROS can induce significant oxidative damage in mitochondria from both normal and tumor tissues and this can be inhibited by natural antioxidants like tocotrienols, nicotinamide and caffeine. Damage, on the other hand, can be enhanced by deuteration of the buffer and oxygenation. Our results hence demonstrated that mitochondria were sensitive to damage by ROS and its modulation may have potential uses in prevention of the disease in normal tissues; if damage can be selectively induced in tumor, it can lead to its regression.