γ-Tocotrienol is a novel inhibitor of constitutive and inducible STAT3 signalling pathway in human hepatocellular carcinoma: potential role as an antiproliferative, pro-apoptotic and chemosensitizing agent

Rajendran P, Li F, Manu KA, Shanmugam MK, Loo SY, Kumar AP, Sethi G.

Br J Pharmacol. 2011 May;163(2):283-98.

Background & Purpose: Activation of signal transducer and activator of transcription 3 (STAT3) play a critical role in the survival, proliferation, angiogenesis and chemoresistance of tumour cells. Thus, agents that suppress STAT3 phosphorylation have potential as cancer therapies. In the present study, we investigated whether the apoptotic, antiproliferative and chemosensitizing effects of γ-tocotrienol are associated with its ability to suppress STAT3 activation in hepatocellular carcinoma (HCC).

Experimental Approach: The effect of γ-tocotrienol on STAT3 activation, associated protein kinases and phosphatase, STAT3-regulated gene products, cellular proliferation and apoptosis in HCC cells was investigated.

Key Results: γ-Tocotrienol inhibited both the constitutive and inducible activation of STAT3 with minimum effect on STAT5. γ-Tocotrienol also inhibited the activation of Src, JAK1 and JAK2 implicated in STAT3 activation. Pervanadate reversed the γ-tocotrienol-induced down-regulation of STAT3, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that γ-tocotrienol induced the expression of the tyrosine phosphatase SHP-1 and deletion of the SHP-1 gene by small interfering RNA abolished the ability of γ-tocotrienol to inhibit STAT3 activation. γ-Tocotrienol also down-regulated the expression of STAT3-regulated gene products, including cyclin D1, Bcl-2, Bcl-xL, survivin, Mcl-1 and vascular endothelial growth factor. Finally, γ-tocotrienol inhibited proliferation, induced apoptosis and significantly potentiated the apoptotic effects of chemotherapeutic drugs (paclitaxel and doxorubicin) used for the treatment of HCC.

Conclusions & Implications: Overall, these results suggest that γ-tocotrienol is a novel blocker of the STAT3 activation pathway, with a potential role in future therapies for HCC and other cancers.

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γ-Tocotrienol induces growth arrest through a novel pathway with TGFβ2 in prostate cancer

Campbell SE, Rudder B, Phillips RB, Whaley SG, Stimmel JB, Leesnitzer LM, Lightner J, Dessus-Babus S, Duffourc M, Stone WL, Menter DG, Newman RA, Yang P, Aggarwal BB, Krishnan K.

Free Radic Biol Med. 2011 May 15;50(10):1344-54. Epub 2011 Feb 16.

Regions along the Mediterranean and in southern Asia have lower prostate cancer incidence compared to the rest of the world. It has been hypothesized that one of the potential contributing factors for this low incidence includes a higher intake of tocotrienols. Here we examine the potential of γ-tocotrienol (GT3) to reduce prostate cancer proliferation and focus on elucidating pathways by which GT3 could exert a growth-inhibitory effect on prostate cancer cells. We find that the γ and δ isoforms of tocotrienol are more effective at inhibiting the growth of prostate cancer cell lines (PC-3 and LNCaP) compared with the γ and δ forms of tocopherol. Knockout of PPAR-γ and GT3 treatment show inhibition of prostate cancer cell growth, through a partially PPAR-γ-dependent mechanism. GT3 treatment increases the levels of the 15-lipoxygenase-2 enzyme, which is responsible for the conversion of arachidonic acid to the PPAR-γ-activating ligand 15-S-hydroxyeicosatrienoic acid. In addition, the latent precursor and the mature forms of TGFβ2 are down-regulated after treatment with GT3, with concomitant disruptions in TGFβ receptor I, SMAD-2, p38, and NF-κB signaling.

Tocotrienols-induced inhibition of platelet thrombus formation and platelet aggregation in stenosed canine coronary arteries

Qureshi AA, Karpen CW, Qureshi N, Papasian CJ, Morrison DC, Folts JD.

Lipids Health Dis. 2011 Apr 14;10:58.

Background: Dietary supplementation with tocotrienols has been shown to decrease the risk of coronary artery disease. Tocotrienols are plant-derived forms of vitamin E, which have potent anti-inflammatory, antioxidant, anticancer, hypocholesterolemic, and neuroprotective properties. Our objective in this study was to determine the extent to which tocotrienols inhibit platelet aggregation and reduce coronary thrombosis, a major risk factor for stroke in humans. The present study was carried out to determine the comparative effects of α-tocopherol, α-tocotrienol, or tocotrienol rich fraction (TRF; a mixture of α-+γ-+δ-tocotrienols) on in vivo platelet thrombosis and ex vivo platelet aggregation (PA) after intravenous injection in anesthetized dogs, by using a mechanically stenosed circumflex coronary artery model (Folts’ cyclic flow model).

Results: Collagen-induced platelet aggregation (PA) in platelet rich plasma (PRP) was decreased markedly after treatment with α-tocotrienol (59%; P<0.001) and TRF (92%; P<0.001). α-Tocopherol treatment was less effective, producing only a 22% (P<0.05) decrease in PA. Adenosine diphosphate-induced (ADP) PA was also decreased after treatment with α-tocotrienol (34%; P<0.05) and TRF (42%; P<0.025). These results also indicate that intravenously administered tocotrienols were significantly better than tocopherols in inhibiting cyclic flow reductions (CFRs), a measure of the acute platelet-mediated thrombus formation. Tocotrienols (TRF) given intravenously (10 mg/kg), abolished CFRs after a mean of 68 min (range 22 -130 min), and this abolition of CFRs was sustained throughout the monitoring period (50-160 min).Next, pharmacokinetic studies were carried out and tocol levels in canine plasma and platelets were measured. As expected, α-Tocopherol treatment increased levels of total tocopherols in post- vs pre-treatment specimens (57 vs 18 μg/mL in plasma, and 42 vs 10 μg/mL in platelets). However, treatment with α-tocopherol resulted in slightly decreased levels of tocotrienols in post- vs pre-treatment samples (1.4 vs 2.9 μg/mL in plasma and 2.3 vs 2.8 μg/mL in platelets). α-Tocotrienoltreatment increased levels of both tocopherols and tocotrienols in post- vs pre-treatment samples (tocopherols, 45 vs 10 μg/mL in plasma and 28 vs 5 μg/mL in platelets; tocotrienols, 2.8 vs 0.9 μg/mL in plasma and 1.28 vs 1.02 μg/mL in platelets). Treatment with tocotrienols (TRF) also increased levels of tocopherols and tocotrienols in post- vs pre-treatment samples (tocopherols, 68 vs 20 μg/mL in plasma and 31.4 vs 7.9 μg/mL in platelets;tocotrienols, 8.6 vs 1.7 μg/mL in plasma and 3.8 vs 3.9 μg/mL in platelets).

Conclusions: The present results indicate that intravenously administered tocotrienols inhibited acute platelet-mediated thrombus formation, and collagen and ADP-induced platelet aggregation. α-Tocotrienols treatment induced increases in α-tocopherol levels of 4-fold and 6-fold in plasma and platelets, respectively. Interestingly, tocotrienols (TRF) treatment induced a less pronounced increase in the levels of tocotrienols in plasma and platelets, suggesting that intravenously administered tocotrienols may be converted to tocopherols. Tocotrienols, given intravenously, could potentially prevent pathological platelet thrombus formation and thus provide a therapeutic benefit in conditions such as stroke and myocardial infarction.

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Gamma tocotrienols protects against radiation exposure

Lisa Hutson

Lisa Hutson

A form of Vitamin E may help protect against high levels of radiation exposure. Studies show that a potent form of Vitamin E called gamma tocotrienols may counteract the harmful effects of radiation. “It is something we have been working on for about five years now,” says Dr. Martin Hauer-Jensen, director of radiation health at UAMS.

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Tocotrienols have potent antifibrogenic effects in human intestinal fibroblasts

Luna J, Masamunt MC, Rickmann M, Mora R, España C, Delgado S, Llach J, Vaquero E, Sans M.

Inflamm Bowel Dis. 2011 Mar;17(3):732-41

Background: Excessive fibroblast expansion and extracellular matrix (ECM) deposition are key events for the development of bowel stenosis in Crohn’s disease (CD) patients. Tocotrienols are vitamin E compounds with proven in vitro antifibrogenic effects on rat pancreatic fibroblasts. We aimed at investigating the effects of tocotrienols on human intestinal fibroblast (HIF) proliferation, apoptosis, autophagy, and synthesis of ECM.

Methods: HIF isolated from CD, ulcerative colitis (UC), and normal intestine were treated with tocotrienol-rich fraction (TRF) from palm oil. HIF proliferation was quantified by (3) H-thymidine incorporation, apoptosis was studied by DNA fragmentation, propidium iodide staining, caspase activation, and poly(ADP-ribose) polymerase cleavage, autophagy was analyzed by quantification of LC3 protein and identification of autophagic vesicles by immunofluorescence and production of ECM components was measured by Western blot.

Results: TRF significantly reduced HIF proliferation and prevented basic fibroblast growth factor-induced proliferation in CD and UC, but not control HIF. TRF enhanced HIF death by promoting apoptosis and autophagy. HIF apoptosis, but not autophagy, was prevented by the pan-caspase inhibitor zVAD-fmk, whereas both types of cell death were prevented when the mitochondrial permeability transition pore was blocked by cyclosporin A, demonstrating a key role of the mitochondria in these processes. TRF diminished procollagen type I and laminin γ-1 production by HIF.

Conclusions: Tocotrienols exert multiple effects on HIF, reducing cell proliferation, enhancing programmed cell death through apoptosis and autophagy, and decreasing ECM production. Considering their in vitro antifibrogenic properties, tocotrienols could be useful to treat or prevent bowel fibrosis in CD patients.

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Tocotrienol-rich fraction prevents cell cycle arrest and elongates telomere length in senescent human diploid fibroblasts

Makpol S, Durani LW, Chua KH, Mohd Yusof YA, Ngah WZ.

J Biomed Biotechnol. 2011;2011:506171. Epub 2011 Mar 30.

This study determined the molecular mechanisms of tocotrienol-rich fraction (TRF) in preventing cellular senescence of human diploid fibroblasts (HDFs). Primary culture of HDFs at various passages were incubated with 0.5 mg/mL TRF for 24 h. Telomere shortening with decreased telomerase activity was observed in senescent HDFs while the levels of damaged DNA and number of cells in G(0)/G(1) phase were increased and S phase cells were decreased. Incubation with TRF reversed the morphology of senescent HDFs to resemble that of young cells with decreased activity of SA-β-gal, damaged DNA, and cells in G(0)/G(1) phase while cells in the S phase were increased. Elongated telomere length and restoration of telomerase activity were observed in TRF-treated senescent HDFs. These findings confirmed the ability of tocotrienol-rich fraction in preventing HDFs cellular ageing by restoring telomere length and telomerase activity, reducing damaged DNA, and reversing cell cycle arrest associated with senescence.

A Regenerative Antioxidant Protocol of Vitamin E and α-Lipoic Acid Ameliorates Cardiovascular and Metabolic Changes in Fructose-Fed Rats

Patel J, Matnor NA, Iyer A, Brown L.

Evid Based Complement Alternat Med. 2011;2011:120801

Type 2 diabetes is a major cause of cardiovascular disease. We have determined whether the metabolic and cardiovascular changes induced by a diet high in fructose in young adult male Wistar rats could be prevented or reversed by chronic intervention with natural antioxidants. We administered a regenerative antioxidant protocol using two natural compounds: α-lipoic acid together with vitamin E (α-tocopherol alone or a tocotrienol-rich fraction), given as either a prevention or reversal protocol in the food. These rats developed glucose intolerance, hypertension, and increased collagen deposition in the heart together with an increased ventricular stiffness. Treatment with a fixed combination of vitamin E (either α-tocopherol or tocotrienol-rich fraction, 0.84 g/kg food) and α-lipoic acid (1.6 g/kg food) normalized glucose tolerance, blood pressure, cardiac collagen deposition, and ventricular stiffness in both prevention and reversal protocols in these fructose-fed rats. These results suggest that adequate antioxidant therapy can both prevent and reverse the metabolic and cardiovascular damage in type 2 diabetes.

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Pentoxifylline enhances the radioprotective properties of γ-tocotrienol: Differential effects on the hematopoietic, gastrointestinal and vascular systems

Berbée M, Fu Q, Garg S, Kulkarni S, Kumar KS, Hauer-Jensen M.

Radiat Res. 2011 Mar;175(3):297-306.

The vitamin E analog γ-tocotrienol (GT3) is a potent radioprotector and mitigator. This study was performed to (a) determine whether the efficacy of GT3 can be enhanced by the addition of the phosphodiesterase inhibitor pentoxifylline (PTX) and (b) to obtain information about the mechanism of action. Mice were injected subcutaneously with vehicle, GT3 [400 mg/kg 24 h before total-body irradiation (TBI)], PTX (200 mg/kg 30 min before TBI), or GT3+PTX before being exposed to 8.5-13 Gy TBI. Overall lethality, survival time and intestinal, hematopoietic and vascular injury were assessed. Cytokine levels in the bone marrow microenvironment were measured, and the requirement for endothelial nitric oxide synthase (eNOS) was studied in eNOS-deficient mice. GT3+PTX significantly improved survival compared to GT3 alone and provided full protection against lethality even after exposure to 12.5 Gy. GT3+PTX improved bone marrow CFUs, spleen colony counts and platelet recovery compared to GT3 alone. GT3 and GT3+PTX increased bone marrow plasma G-CSF levels as well as the availability of IL-1α, IL-6 and IL-9 in the early postirradiation phase. GT3 and GT3+PTX were equally effective in ameliorating intestinal injury and vascular peroxynitrite production. Survival studies in eNOS-deficient mice and appropriate controls revealed that eNOS was not required for protection against lethality after TBI. Combined treatment with GT3 and PTX increased postirradiation survival over that with GT3 alone by a mechanism that may depend on induction of hematopoietic stimuli. GT3+PTX did not reduce GI toxicity or vascular oxidative stress compared to GT3 alone. The radioprotective effect of either drug alone or both drugs in combination does not require the presence of eNOS.

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Reduction of radiation-induced vascular nitrosative stress by the vitamin E analog γ-tocotrienol: Evidence of a role for tetrahydrobiopterin

Berbee M, Fu Q, Boerma M, Pathak R, Zhou D, Kumar KS, Hauer-Jensen M.

Int J Radiat Oncol Biol Phys. 2011 Mar 1;79(3):884-91

Purpose: The vitamin E analog γ-tocotrienol (GT3) is a powerful radioprotector. GT3 reduces postradiation vascular peroxynitrite production, an effect dependent on inhibition of hydroxy-methylglutaryl-coenzyme A reductase. Hydroxy-methylglutaryl-coenzyme A reductase inhibitors mediate their pleiotropic effects via endothelial nitric oxide synthase that requires the cofactor tetrahydrobiopterin (BH4). This study investigated the effects of radiation on BH4 bioavailability and of GT3 on BH4 metabolism.

Methods And Materials: Mice were exposed to 8.5 Gy of total body irradiation (TBI). Lung BH4 and total biopterin concentrations were measured 0, 3.5, 7, 14, and 21 days after TBI by use of differential oxidation followed by high-performance liquid chromatography. The effect of exogenous GT3 and BH4 treatment on postradiation vascular oxidative stress and bone marrow colony-forming units were assessed in vivo. The effect of GT3 on endothelial cell apoptosis and endothelial expression of guanosine triphosphate (GTP) cyclohydrolase 1 (GTPCH), GTPCH feedback regulatory protein (GFRP), GFRP transcription, GFRP protein levels, and GFRP-GTPCH protein binding was determined in vitro.

Results: Compared with baseline levels, lung BH4 concentrations decreased by 24% at 3.5 days after TBI, an effect that was reversed by GT3. At 14 and 21 days after TBI, compensatory increases in BH4 (58% and 80%, respectively) were observed. Relative to vehicle-treated controls, both GT3 and BH4 supplementation reduced postirradiation vascular peroxynitrite production at 3.5 days (by 66% and 33%, respectively), and BH4 resulted in a 68% increase in bone marrow colony-forming units. GT3 ameliorated endothelial cell apoptosis and reduced endothelial GFRP protein levels and GFRP-GTPCH binding by decreasing transcription of the GFRP gene.

Conclusions: BH4 bioavailability is reduced in the early postradiation phase. Exogenous administration of BH4 reduces postirradiation vascular oxidative stress. GT3 potently reduces the expression of GFRP, one of the key regulatory proteins in the BH4 pathway, and may thus exert some of its beneficial effects on postradiation free radical production partly by counteracting the decrease in BH4.

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δ-Tocotrienol and quercetin reduce serum levels of nitric oxide and lipid parameters in female chickens

Qureshi AA, Reis JC, Qureshi N, Papasian CJ, Morrison DC, Schaefer DM.

Lipids Health Dis. 2011 Feb 28;10:39.

Background: Chronic, low-grade inflammation provides a link between normal ageing and the pathogenesis of age-related diseases. A series of in vitro tests confirmed the strong anti-inflammatory activities of known inhibitors of NF-κB activation (δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride, and dexamethasone). δ-Tocotrienol also suppresses β-hydroxy-β-methylglutaryl coenzyme A (HMG-CoA) reductase activity (the rate-limiting step in de novo cholesterol synthesis), and concomitantly lowers serum total and LDL cholesterol levels. We evaluated these compounds in an avian model anticipating that a dietary additive combining δ-tocotrienol with quercetin, riboflavin, (-) Corey lactone, amiloride, or dexamethasone would yield greater reductions in serum levels of total cholesterol, LDL-cholesterol and inflammatory markers (tumor necrosis factor-α [TNF-α], and nitric oxide [NO]), than that attained with the individual compounds.

Results: The present results showed that supplementation of control diets with all compounds tested except riboflavin, (-) Corey lactone, and dexamethasone produced small but significant reductions in body weight gains as compared to control. (-) Corey lactone and riboflavin did not significantly impact body weight gains. Dexamethasone significantly and markedly reduced weight gain (>75%) compared to control. The serum levels of TNF-α and NO were decreased 61% – 84% (P < 0.001), and 14% – 67%, respectively, in chickens fed diets supplemented with δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride, or dexamethasone as compared to controls. Significant decreases in the levels of serum total and LDL-cholesterol were attained with δ-tocotrienol, quercetin, riboflavin and (-) Corey lactone (13% – 57%; P < 0.05), whereas, these levels were 2-fold higher in dexamethasone treated chickens as compared to controls. Parallel responses on hepatic lipid infiltration were confirmed by histological analyses. Treatments combining δ-tocotrienol with the other compounds yielded values that were lower than individual values attained with either δ-tocotrienol or the second compound. Exceptions were the significantly lower total and LDL cholesterol and triglyceride values attained with the δ-tocotrienol/(-) Corey lactone treatment and the significantly lower triglyceride value attained with the δ-tocotrienol/riboflavin treatment. δ-Tocotrienol attenuated the lipid-elevating impact of dexamethasone and potentiated the triglyceride lowering impact of riboflavin. Microarray analyses of liver samples identified 62 genes whose expressions were either up-regulated or down-regulated by all compounds suggesting common impact on serum TNF-α and NO levels. The microarray analyses further identified 41 genes whose expression was differentially impacted by the compounds shown to lower serum lipid levels and dexamethasone, associated with markedly elevated serum lipids.

Conclusion: This is the first report describing the anti-inflammatory effects of δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride, and dexamethasone on serum TNF-δ and NO levels. Serum TNF-δ levels were decreased by >60% by each of the experimental compounds. Additionally, all the treatments except with dexamethasone, resulted in lower serum total cholesterol, LDL-cholesterol and triglyceride levels. The impact of above mentioned compounds on the factors evaluated herein was increased when combined with δ-tocotrienol.

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