The effects of tocotrienol-rich fraction (TRF), α-tocopherol (T) and α-tocopheryl acetate (TA) on lipopolysaccharide (LPS)-induced inflammatory responses in mouse peritoneal macrophages were examined. Results showed that at 5-30μg/ml, all test compounds plus 1μg/ml LPS exhibited no cytotoxic effects on macrophage cells. Compared with T and TA, TRF showed the strongest anti-inflammatory activity as demonstrated by its potency in inhibiting the LPS-induced nitric oxide (NO), prostaglandin E(2) (PGE(2)), and proinflammatory cytokine (TNF-α, IFN-γ, IL-1β and IL-6) production. At 10μg/ml, it significantly blocked the LPS induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, but has no effect on cyclooxygenase-1 (COX-1). Furthermore, TRF also showed a greater inhibition on the nuclear factor kappa B (NF-κB) expression than T and TA. These results suggest that TRF could be a better agent than T and TA for use in the prevention of chronic inflammatory diseases.
Publications
A key regulator of cholesterol homeostasis, SREBP-2, can be targeted in prostate cancer cells with natural products
James R. Krycer, Lisa Phan, Andrew J. Brown
Biochem J. 2012 Sep 1;446(2):191-201
There is growing evidence showing prostate cancer cells have perturbed cholesterol homeostasis, accumulating cholesterol to promote cell-growth. Consequently, cholesterol lowering drugs like statins are being evaluated in prostate cancer treatment. Furthermore, natural products such as betulin (from birch tree bark) and tocotrienol (a minor form of Vitamin E) have been shown to lower cholesterol levels. Using these drugs and oxysterols, we determine which aspects of cholesterol homeostasis should be targeted in prostate cancer –e.g., cellular cholesterol levels are increased by the transcription factor Sterol-Regulatory Element Binding Protein isoform 2 (SREBP-2), whereas the Liver X Receptor (LXR) promotes cholesterol efflux. Whilst betulin exerted non-specific effects on cell viability, tocotrienols produced a strong direct correlation between SREBP-2 activity and cell viability. Mechanistically, tocotrienols lowered SREBP-2 activity by degrading mature SREBP-2 independently of the proteasome. In contrast, no correlation was seen between LXR activity and cell viability, implying SREBP-2 is a better target than LXR for prostate cancer treatment. Lastly, androgen-dependent and -independent LNCaP cells were both sensitive to tocotrienols. Overall, this suggests that tocotrienols and other drugs targeting the SREBP-2 pathway are a potential therapeutic option for prostate cancer.
Inhibition of nitric oxide and inflammatory cytokines in LPS-stimulated murine macrophages by resveratrol, a potent proteasome inhibitor
Qureshi AA, Guan XQ, Reis JC, Papasian CJ, Jabre S, Morrison DC, Qureshi N.
Lipids Health Dis. 2012 Jul 10;11:76.
Background: Altered immune function during ageing results in increased production of nitric oxide (NO) and other inflammatory mediators. Recently, we have reported that NO production was inhibited by naturally-occurring proteasome inhibitors (quercetin, δ-tocotrienol, and riboflavin) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and thioglycolate-elicited peritoneal macrophages from C57BL/6 mice. In a continuous effort to find more potent, non-toxic, commercially available, naturally-occurring proteasome inhibitors that suppress inflammation, the present study was carried out to describe the inhibition of NF-κB activation and NO, TNF-α, IL-6, IL-1β, and iNOS expression by trans-resveratrol, trans-pterostilbene, morin hydrate, and nicotinic acid in LPS-induced RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice.
Results: The present results indicate that resveratrol, pterostilbene, and morin hydrate caused significant inhibition (>70% to 90%; P < 0.02) in the activities of chymotrypsin-like, trypsin-like, and post-acidic (post-glutamase) proteasome sites in RAW 264.7 cells at a dose of only 20 μM. These compounds also inhibited the production of NO by RAW-264.7 cells stimulated with LPS alone (>40%; P < 0.05), or LPS + interferon-γ (IFN-γ; >60%; P < 0.02). Furthermore, resveratrol, pterostilbene, morin hydrate, and quercetin suppressed secretion of TNF-α (>40%; P < 0.05) in LPS-stimulated RAW 264.7 cells, and suppressed NF-κB activation (22% to 45%; P < 0.05) in LPS-stimulated HEK293T cells. These compounds also significantly suppressed LPS-induced expression of TNF-α, IL-1β, IL-6, and iNOS genes in RAW 264.7 cells, and also in thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice.
Conclusions: The present results clearly demonstrate that resveratrol and pterostilbene are particularly potent proteasome inhibitors that suppress expression of genes, and production of inflammatory products in LPS-stimulated RAW 264.7 cells, and macrophages from C57BL/6 and BALB/c mice. Resveratrol and pterostilbene which are present in grapes, blueberries, and red wine, have been implicated as contributing factors to the lower incidence of cardiovascular disease in the French population, despite their relatively high dietary fat intake. Consequently, it appears likely that the beneficial nutritional effects of resveratrol and pterostilbene are due at least in part, to their ability to inhibit NF-κB activation by the proteasome, thereby suppressing activation of pro-inflammatory cytokines and iNOS genes, resulting in decreased secretion of TNF-α, IL-1β, IL-6, and NO levels, in response to inflammatory stimuli. This is the first report demonstrating that resveratrol and pterostilbene act as proteasome inhibitors, thus providing a mechanism for their anti-inflammatory effects.
γ-Tocotrienol inhibits cell viability through suppression of β-catenin/Tcf signaling in human colon carcinoma HT-29 cells
Xu W, Du M, Zhao Y, Wang Q, Sun W, Chen B.
J Nutr Biochem. 2012 Jul;23(7):800-7. Epub 2011 Aug 17.
γ-Tocotrienol, a major component of the tocotrienol-rich fraction of palm oil, has been suggested to have antioxidant and anticancer activity as well as potent chemopreventive effects on tumor cells. In this study, the mechanisms underlying γ-tocotrienol-mediated growth inhibition of human carcinoma HT-29 cells were further investigated, especially in correlation with the involvement of β-catenin/T-cell factor (Tcf) signaling pathway. We found that γ-tocotrienol could strongly suppress the transcriptional activity of β-catenin/Tcf signaling pathway in HT-29 cells. γ-Tocotrienol inhibited the expression level of total β-catenin protein but did not significantly affect the phosphorylated β-catenin level. Meanwhile, γ-tocotrienol down-regulated the protein level of nuclear β-catenin and induced its redistribution to cell membrane. Furthermore, γ-tocotrienol suppressed the expression of downstream target genes such as c-myc, cyclin D1 and survivin. The results demonstrated that γ-tocotrienol-inhibited growth and -induced apoptosis in HT-29 cells were accompanied by significant inhibition of β-catenin/Tcf signaling. Blocking the expression of β-catenin with small interfering RNA significantly suppressed the ability of γ-tocotrienol to reduce viability and induce apoptosis in HT-29 cells. Thus, our data suggested that γ-tocotrienol exerts its anticancer activity through β-catenin/Tcf signaling, and β-catenin is a target for γ-tocotrienol in the Wnt/β-catenin signaling pathway.
Delta-tocotrienol augments cisplatin-induced suppression of non-small cell lung cancer cells via inhibition of the Notch-1 pathway
Ji, X., Wang, Z., Sarkar, F. H., Gupta, S. V.
Anticancer Res. 2012 Jul;32(7):2647-55.
Non-small cell lung cancer (NSCLC), accounting for 80% of lung cancers, is the leading cause of all cancer deaths. Previously, we demonstrated that delta-tocotrienol inhibits NSCLC cell proliferation, invasion and induces apoptosis by down-regulation of the Notch-1 signaling pathway. The objective of this study was to investigate whether delta-tocotrienol, could enhance the anticancer effects of cisplatin. Treatment with a combination of delta-tocotrienol and cisplatin resulted in a dose-dependent, significant inhibition of cell growth, migration, invasiveness, and induction of apoptosis in NSCLC cells, as compared to the single agents. This was associated with a decrease in NF-kappaB DNA binding activity, decrease in Notch-1, Hes-1, Bcl-2 and increase in cleaved Caspase-3 and PARP expressions. These results suggest that down-regulation of Notch-1, via inhibition of NF-kappaB signaling pathways by delta-tocotrienol and cisplatin, in combination, could provide a potential novel approach for tumor arrest in NSCLC, while lowering the effective dose of cisplatin.
Suppression of Nitric Oxide Production and Cardiovascular Risk Factors in Healthy Seniors and Hypercholesterolemic Subjects by a Combination of Polyphenols and Vitamins
Qureshi AA, Khan DA, Mahjabeen W, Papasian CJ, Qureshi N.
J Clin Exp Cardiolog. 2012 Jun 7;S5:8.
Background: Dysregulated immune function associated with ageing has been implicated in a variety of human diseases. We have demonstrated the anti-inflammatory properties of resveratrol, pterostilbene, morin hydrate, quercetin, δ-tocotrienol, riboflavinin a variety of experimental animal models, and determined that these compounds act by inhibiting proteasome activity.
Aims: To determine whether serum nitric oxide (NO) levels increase with age in humans, and whether the combined cholesterol-lowering and inflammation-reducing properties of resveratrol, pterostilbene, Morin hydrate, quercetin, δ-tocotrienol, riboflavin, and nicotinic acid would reduce cardiovascular risk factors in humans when used as nutritional supplements with, or without, other dietary changes.
Methods: Elderly human subjects were stratified into two groups based on total serum cholesterol levels. Initial total serum cholesterol levels were normal and elevated in Group 1 and 2 subjects, respectively. Baseline serum NO, C-reactive protein (CRP), γ-glutamyltransferase (γ-GT) activity, uric acid, total antioxidant status (TAS), total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides levels were established over a four week period. Group 1 subjects subsequently received nutritional supplementation with one of two different combinations (NS-7 = 25 mg of each, resveratrol, pterostilbene, quercetin, δ-tocotrienol, nicotinic acid, morin hydrate or NS-6 = morin hydrate replaced with quercetin, 50 mg/capsule). Group 2 subjects also received these nutritional supplements (two capsules/d), but an AHA Step-1 diet was also implemented. After these interventions were administered for four weeks, the above parameters were re-measured and changes from baseline levels determined. Nitric acid (NO) levels in children, young adults, and seniors were also compared.
Results: The key results of the current study were: 1) that serum NO levels were significantly increased in seniors compared to both children (~80%) and young adults (~65%); 2) that the intake of two capsules/d of NS-7 or NS-6 for four weeks significantly (P < 0.05) decreased serum NO (39%, 24%), CRP (19%, 21%), uric acid (6%, 12%) levels, and γ-GT activity (8%, 6%), respectively in free-living healthy seniors; 3) that serum NO (36%, 29%), CRP (29%, 20%), uric acid (6%, 9%) γ-GT activity (9%, 18%), total cholesterol (8%, 11%), LDL-cholesterol (10%, 13%), and triglycerides (16%, 23%) levels were significantly (P < 0.02) decreased in hypercholesterolemic subjects restricted to AHA Step-1 diet plus intake of SN-7 or SN-6 (two capsules/d), respectively; 4) that TAS was increased (3%, 9%; P < 0.05) in free-living healthy seniors receiving NS-7 or NS-6 alone, and in hypercholesterolemic subjects plus AHA Step-1 diet (20%, 12%; P < 0.02) with either of the combinations tested.
Conclusion: Serum NO levels are elevated in elderly humans compared to children or young adults. Diet supplementation with combinations of resveratrol, pterostilbene, morin hydrate, quercetin, δ-tocotrienol, riboflavin, and nicotinic acid reduce cardiovascular risk factors in humans when used as nutritional supplements with, or without, other dietary changes.
Redox-inactive analogue of tocotrienol as a potential anti-cancer agent
Yano T, Sato A, Sekine M, Virgona N, Ota M. Source
Anticancer Agents Med Chem. 2012 Jun 18. [Epub ahead of print]
Vitamins are prominent among natural or endogenous compounds that are considered to be beneficial for both prevention and therapy of various human ailments. The vitamin E group of compounds composed of tocopherol and tocotrienol isoforms, has been subsequently proven to have health benefits including antioxidant and related protective properties. However, individual isoforms exhibit a wide-range of antioxidant potencies.Tocotrienol (T3) displays powerful anticancer activity that is often not exhibited by tocopherols, by modulating multiple intracellular signaling pathways associated with tumor cell proliferation and survival. The anticancer effect of T3 remains not fully understood but generally is mediated independently of its antioxidant activity. Further we have synthesized a new redox-inactive analogue of T3, 6-O-carboxypropyl-α-tocotrienol (T3E) showing considerable promise for stronger anticancer potency than its mother compound. In this mini-review, we particularly focus upon the anticancer action of the above active components of vitamin E and describe current research on the anticancer effects of T3 irrespective of antioxidant activity.
Delta- and gamma-tocotrienols induce classical ultrastructural apoptotic changes in human T lymphoblastic leukemic cells
Rebecca S.Y. Wong, Ammu K. Radhakrishnan,2 Tengku Azmi Tengku Ibrahim, and Soon-Keng Cheong
Microsc Microanal. 2012 Jun;18(3):462-9.
Tocotrienols are isomers of the vitamin E family, which have been reported to exert cytotoxic effects in various cancer cells. Although there have been some reports on the effects of tocotrienols in leukemic cells, ultrastructural evidence of tocotrienol-induced apoptotic cell death in leukemic cells is lacking. The present study investigated the effects of three isomers of tocotrienols (alpha, delta, and gamma) on a human T lymphoblastic leukemic cell line (CEM-SS). Cell viability assays showed that all three isomers had cytotoxic effects ( p , 0.05) on CEM-SS cells with delta-tocotrienol being the most potent. Transmission electron microscopy showed that the cytotoxic effects by delta- and gamma-tocotrienols were through the induction of an apoptotic pathway as demonstrated by the classical ultrastructural apoptotic changes characterized by peripheral nuclear chromatin condensation and nuclear fragmentation. These findings were confirmed biochemically by the demonstration of phosphatidylserine externalization via flow cytometry analysis. This is the first study showing classical ultrastructural apoptotic changes induced by delta- and gamma-tocotrienols in human T lymphoblastic leukemic cells.
Distinct roles of different forms of vitamin E in DHA-induced apoptosis in triple-negative breast cancer cells
Ailian Xiong, Weiping Yu, Richa Tiwary, Bob G. Sanders and Kimberly Kline
Mol Nutr Food Res. 2012 Jun;56(6):923-34
Scope: Docosahexaenoic acid (DHA) has been shown to exhibit anticancer actions in vitro and in vivo in a variety of cancers. Here, we investigated the role for DHA in inducing apoptosis in triple-negative breast cancer (TNBC) and studied the mechanisms of action.
Methods and Results: DHA induces apoptosis as detected byAnnexinV-FITC/PI assay as well as induces cleavage of caspase-8 and -9, endoplasmic reticulum stress (ERS), and elevated levels of death receptor-5 (DR5) protein expression as detected by western blot assays. Chemical inhibitors of caspase-8 and -9 and small interfering RNAs (siRNAs) show DHA to induce ERS/CHOP/DR5-mediated caspase-8 and -9 dependent apoptosis. Furthermore, DHA induces elevated cellular levels of reactive oxygen species (ROS) and antioxidant; RRR-α-tocopherol (_T) blocked DHA-induced apoptotic events. In contrast to the antagonistic impact of αT, gamma tocotrienol(γT3) was demonstrated to cooperate with DHA in inducing apoptotic events in TNBC cells.
Conclusion: Data, for the first time, demonstrate thatDHAinduces apoptosis in TNBC cells via activation of ERS/CHOP/DR5-mediated caspase-8 and -9 dependent pro-apoptotic events, and that different forms of vitamin E exhibit distinct effects on DHA-induced apoptosis; namely, inhibition by αT and enhancement by γT3.
Mechanism of radioprotection by δ-tocotrienol: Pharmacokinetics, pharmacodynamics and modulation of signalling pathways
Satyamitra M, Ney P, Graves J, Mullaney C, Srinivasan V.
Br J Radiol. 2012 Jun 6.
Objective: To investigate the correlation between in vivo δ-tocotrienol (DT3) pharmacokinetcs, pharmacodynamics and radiation protection, and to evaluate the effect of DT3 pre-treatment on radiation-induced alterations in apoptotic and autophagic pathways.Methods: We evaluated pharmacokinetics (plasma, 0.5 to 12 h) and pharmacodynamics (peripheral blood indices, day 3, 7, 10 and 14) after a single subcutaneous injection of 300 mg kg DT3 in unirradiated CD2F1 mice. Next, we monitored 30-day post-irradiation survival (9.25 Gy) and haematopoietic recovery of DT3-treated mice (7 Gy) exposed to cobalt-60 γ irradiation. The effects of DT3 on irradiated bone marrow apoptosis and autophagy were determined by analyses of key caspases (3, 7, 9 and 8), beclin-1 and LC3 conversion.Results: Plasma concentration of DT3 reached ∼195 µM (Cmax) 1 h after injection (Tmax), and DT3 was eliminated from plasma 12 h later. In unirradiated mice, DT3 significantly increased white blood cells (WBCs), neutrophils, lymphocytes (day 3 post-DT3 injection) and platelets (day 7) by 1.5-2-fold, over vehicle-treated control. DT3 pre-treatment improved 30-day survival to 100% (∼15% in control) and accelerated recovery of reticulocytes, platelets, WBCs, neutrophils, lymphocytes and monocytes in peripheral blood. DT3 reduced activation of caspase-8, caspases-3 and -7, inherent to apoptosis, while increasing autophagy-related beclin-1 expression in irradiated bone marrow.Conclusion: These data indicate that DT3 stimulates multilineage haematopoiesis, protects against radiation-induced apoptosis downstream of the mitochondria and stimulates cytoprotective autophagy. Apart from a potent antioxidant activity, DT3 may elicit survival advantage following irradiation by enhancing haematopoiesis and modulating signalling pathways.