Metabolism of 11'-α- and 11'-γ-Tocomonoenols in HepG2 Cells Favors the γ-Congener and Results Predominantly in Carboxymethylbutyl-Hydroxychromans.

SCOPE: Tocomonoenols (T1) are little-known vitamin E derivatives naturally occurring in foods. Limited knowledge exists regarding the cellular uptake and metabolism of α-tocomonoenol (αT1) and none about that of γ-tocomonoenol (γT1). METHODS AND RESULTS: The study investigates the cytotoxicity, uptake, and metabolism of αT1 and γT1 in HepG2 cells compared to the α- and γ-tocopherols (T) and -tocotrienols (T3). None of the studied tocochromanols are cytotoxic up to 100 µmol L-1. The uptake of the γ-congeners is significantly higher than that of the corresponding α-forms, whereas no significant differences are observed based on the degree of saturation of the sidechain. Carboxymethylbutyl-hydroxychromans (CMBHC) are the predominant short-chain metabolites of all tocochromanols and conversion is higher for γT1 than αT1 as well as for the γ-congeners of T and T3. The rate of metabolism increases with the number of double bonds in the sidechain. The rate of metabolic conversion of the T1 is more similar to tocopherols than to that of the tocotrienols. CONCLUSION: This is the first evidence that both αT1 and γT1 follow the same sidechain degradation pathway and exert similar rates of metabolism than tocopherols. Therefore, investigation into the biological activities of tocomonoenols is warranted.

Enzyme-treated red rice (Oryza sativa L.) bran extracts mitigate inflammatory markers in RAW 264.7 macrophage cells and exhibit anti-inflammatory efficacy greater/comparable to ferulic acid, catechin, γ-tocopherol, and γ-oryzanol.

ETHNOPHARMACOLOGICAL RELEVANCE: Rice (Oryza sativa L.), a staple food for a significant portion of the global population, has been recognized for its traditional medicinal properties for centuries. Rice bran, a by-product of rice milling, contains many bioactive compounds with potential pharmaceutical and therapeutic benefits. In recent years, research has highlighted the anti-inflammatory potential of rice bran, contributed by the bioactive components concentrated in their bran but, unfortunately, entrapped in the bran matrix, with limited bioavailability. Previous studies have reported that the enzymatic treatment of rice bran improves the bran's bioactive compound profile but did not investigate its impact on chronic conditions such as inflammation. AIM OF THE STUDY: This study investigates the anti-inflammatory effects of endo-1,4-ß-xylanase (ERB) and Viscozyme (VRB) treated red rice bran extracts against lipopolysaccharide-induced inflammation in RAW264.7 macrophages in comparison with non-enzyme-treated bran (CRB). Further established their efficacy with known anti-inflammatory compounds-ferulic acid (FA), catechin (CAT), γ-tocopherol (GTP), and γ-oryzanol (ORZ). MATERIALS AND METHODS: The RAW 264.7 macrophage cells were pre-treated with non-toxic concentrations (10-200 µg/mL) of FA, CAT, GTP, ORZ, CRB, ERB, and VRB, followed by inflammatory stimulation with LPS for 24 h. Further, the cell supernatant and pellets were harvested to study the anti-inflammatory effects by evaluating and measuring their efficacy in inhibiting pro-inflammatory cytokines (TNF-α, IL-6, IL-10, IL-1ß) and mediators (ROS, NO, PGE2, COX2, iNOS) through biochemical, ELISA, and mRNA expression studies. RESULTS: The findings showed that both ERB and VRB effectively inhibited the production of pro-inflammatory markers (TNF-α, IL-6) and mediators (ROS, NO, PGE2) by downregulating mRNA expressions of inflammatory genes (TNF-α, IL-1ß, IL-6, IL-10, COX2, iNOS) and demonstrated anti-inflammatory efficacy higher than CRB. On comparison, ERB demonstrated exceptional efficacy by causing a reduction of 48% in ROS, 20% in TNF-α, and 23% in PGE2 at 10 µg/mL, surpassing the anti-inflammatory capabilities of all the bioactive compounds, FA and ORZ, respectively. At the same time, VRB exhibited remarkable efficacy by reducing NO production by 52% at 200 µg/mL and IL-6 by 66% at 10 µg/mL, surpassing FA, CAT, ORZ, and GTP. Further, ERB downregulated the mRNA expression of IL-10 and iNOS, while VRB downregulated TNF-α, IL-1ß, and COX2 expression. Both extracts equally downregulated IL-6 expression at 10 µg/mL, demonstrating the efficacy more remarkable/on par with established anti-inflammatory compounds. CONCLUSIONS: Overall, enzyme-treated rice bran/extract, particularly ERB, possesses excellent anti-inflammatory properties, making them promising agents for alternatives to contemporary nutraceuticals/functional food against inflammatory diseases.

Ameliorative effect of oregano (Origanum vulgare) versus silymarin in experimentally induced hepatic encephalopathy.

Hepatic encephalopathy (HE) is a deterioration of brain function in patients suffering from chronic liver disease, cirrhosis as a result of elevated blood ammonia and the production of pseudo-neurotransmitters. Herein, we investigated the chemical composition of hexane extract from Origanum vulgare (O. vulgare) leaves as well as its possible protective effects against thioacetamide (TAA)-induced HE in rats. GC-MS analysis of the extract revealed tentative identification of twenty-five compounds (82.93%), predominated by cholesten-3-one (27.30%), followed by γ-tocopherol (13.52%), α-tocopherol (5.01%), ß-amyrin (5.24%) and α-amyrin (4.89%). Albino rats were distributed into seven groups (n = 7). G1 served as negative control; G2 and G3 served as controls treated with O. vulgare (100 and 200 mg/kg/p.o b.w, respectively); G4 served as TAA-positive control group (100 mg/kg/day/i.p., three alternative days per week for six weeks); G5, G6, and G7 served as TAA -induced HE rat model that received O. vulgare 100, O. vulgare 200, and silymarin (100 mg/kg of SILY, as standard drug), respectively. TAA showed depressive and anxiety-like behaviors in forced swimming test (FST) and reduction of cognitive score in elevated plus-maze test (EPMT) as well as impairment of locomotor and exploratory activities in open-field test (OFT). TAA caused a significant decline in body weight gain; however, the relative liver weight and brain water content were statistically increased. TAA-intoxicated rats showed significant increase of serum biomarker enzymes, proinflammatory cytokines, blood ammonia levels, brain serotonin, acetyl cholinesterase and cellular lipid peroxidation with significant decrease of brain dopamine, norepinephrine, antioxidant status. The hepatoprotective/neuro-protective activities of O. vulgare was found to be comparable with that of SILY in HE rats model. Where, treatment of TAA-intoxicated rats with O. vulgare attenuated anxiety, depressive-related behaviors, and reduced the biochemical changes in HE-induced by TAA. Therefore, O. vulgare could be an excellent hepato-/neuroprotective against hepatic injury and HE via improving the oxidative/inflammatory status through its antioxidant and neuro-modulatory properties and its effect is equal to that of SILY.

Network Pharmacology Study on Morus alba L. Leaves: Pivotal Functions of Bioactives on RAS Signaling Pathway and Its Associated Target Proteins against Gout.

M. alba L. is a valuable nutraceutical plant rich in potential bioactive compounds with promising anti-gouty arthritis. Here, we have explored bioactives, signaling pathways, and key proteins underlying the anti-gout activity of M. alba L. leaves for the first-time utilizing network pharmacology. Bioactives in M. alba L. leaves were detected through GC-MS (Gas Chromatography-Mass Spectrum) analysis and filtered by Lipinski's rule. Target proteins connected to the filtered compounds and gout were selected from public databases. The overlapping target proteins between bioactives-interacted target proteins and gout-targeted proteins were identified using a Venn diagram. Bioactives-Proteins interactive networking for gout was analyzed to identify potential ligand-target and visualized the rich factor on the R package via the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway on STRING. Finally, a molecular docking test (MDT) between bioactives and target proteins was analyzed via AutoDock Vina. Gene Set Enrichment Analysis (GSEA) demonstrated that mechanisms of M. alba L. leaves against gout were connected to 17 signaling pathways on 26 compounds. AKT1 (AKT Serine/Threonine Kinase 1), γ-Tocopherol, and RAS signaling pathway were selected as a hub target, a key bioactive, and a hub signaling pathway, respectively. Furthermore, three main compounds (γ-Tocopherol, 4-Dehydroxy-N-(4,5-methylenedioxy-2-nitrobenzylidene) tyramine, and Lanosterol acetate) and three key target proteins-AKT1, PRKCA, and PLA2G2A associated with the RAS signaling pathway were noted for their highest affinity on MDT. The identified three key bioactives in M. alba L. leaves might contribute to recovering gouty condition by inactivating the RAS signaling pathway.

Reduced infiltration of regulatory T cells in tumours from mice fed daily with gamma-tocotrienol supplementation.

Gamma-tocotrienol (γT3) is an analogue of vitamin E with beneficial effects on the immune system, including immune-modulatory properties. This study reports the immune-modulatory effects of daily supplementation of γT3 on host T helper (Th) and T regulatory cell (Treg ) populations in a syngeneic mouse model of breast cancer. Female BALB/c mice were fed with either γT3 or vehicle (soy oil) for 2 weeks via oral gavage before they were inoculated with syngeneic 4T1 mouse mammary cancer cells (4T1 cells). Supplementation continued until the mice were euthanized. Mice (n = 6) were euthanized at specified time-points for various analysis (blood leucocyte, cytokine production and immunohistochemistry). Tumour volume was measured once every 7 days. Gene expression studies were carried out on tumour-specific T lymphocytes isolated from splenic cultures. Supplementation with γT3 increased CD4+ (p < 0.05), CD8+ (p < 0.05) T-cells and natural killer cells (p < 0.05) but suppressed Treg cells (p < 0.05) in peripheral blood when compared to animals fed with the vehicle. Higher interferon (IFN)-γ and lower transforming growth factor (TGF)-ꞵ levels were noted in the γT3 fed mice. Immunohistochemistry findings revealed higher infiltration of CD4+ cells, increased expression of interleukin-12 receptor-beta-2 (IL-12ꞵ2R), interleukin (IL)-24 and reduced expression of cells that express the forkhead box P3 (FoxP3) in tumours from the γT3-fed animals. Gene expression studies showed the down-regulation of seven prominent genes in splenic CD4+ T cells isolated from γT3-fed mice. Supplementation with γT3 from palm oil-induced T cell-dependent cell-mediated immune responses and suppressed T cells in the tumour microenvironment in a syngeneic mouse model of breast cancer.

Dietary alpha- and gamma-tocopherol (1:5 ratio) supplementation attenuates adipose tissue expansion, hepatic steatosis, and expression of inflammatory markers in a high-fat-diet-fed murine model.

OBJECTIVES: The aim of this study was to evaluate the effect of the dietary supplementation of an alpha- and gamma-tocopherol mixture (1:5 ratio) in the adipose tissue expansion, hepatic steatosis, and expression of inflammatory markers induced by consumption of a high-fat diet (HFD) in mice. METHODS: Male C57BL/6 J mice were fed for 12 wk and divided into the following: 1) control diet (CD; 10% fat, 20% protein, 70% carbohydrates); 2) CD + TF (CD plus alpha-tocopherol: 0.7 mg/kg/d, gamma-tocopherol: 3.5 mg/kg/d); 3) HFD (60% fat, 20% protein, 20% carbohydrates); and 4) HFD + TF (HFD plus alpha-tocopherol: 0.7 mg/kg/d, gamma-tocopherol: 3.5 mg/kg/d). General parameters, adipocyte size, liver steatosis, adipose and hepatic tumor necrosis factor-α (TNF-α) and interleukin-1 ß (IL-1ß) expression, hepatic nuclear factor kappa B (NF-κB), and peroxisome proliferator-activated receptor α (PPAR-α) levels were evaluated. RESULTS: Tocopherol supplementation in HFD-fed mice showed a significant decrease in the body weight (19%) and adipose tissue weight (52%), adipose tissue/body weight ratio (36%), and serum triacylglycerols (56%); a 42% decrease (P < 0.05) of adipocyte size compared to HFD; attenuation of liver steatosis by decreasing (P < 0.05) lipid vesicles presence (90%) and total lipid content (75%); and downregulation of inflammatory markers (TNF-α and IL-1ß), along with an upregulation of hepatic PPAR-α expression and its downstream-regulated genes (ACOX and CAT-1), and an inhibition of hepatic NF-κB activation. CONCLUSION: The present study suggests that alpha- and gamma-tocopherol (1:5 ratio) supplementation attenuates the adipocyte enlargement, hepatic steatosis, and metabolic inflammation induced by HFD in association with PPAR-α/NF-κB modulation.

Novel and Predictive QSAR Model for Steroidal and Nonsteroidal 5α- Reductase Type II Inhibitors.

AIMS AND OBJECTIVE: In this study, a novel quantitative structure activity relationship (QSAR) model has been developed for inhibitors of human 5-alpha reductase type II, which are used to treat benign prostate hypertrophy (BPH). METHODS: The dataset consisted of 113 compounds-mainly nonsteroidal-with known inhibitory concentration. Then 3D structures of compounds were optimized and molecular structure descriptors were calculated. The stepwise multiple linear regression was used to select descriptors encoding the inhibitory activity of the compounds. Multiple linear regression (MLR) was used to build up the linear QSAR model. RESULTS: The results obtained revealed that the descriptors which best describe the activity were atom type electropological state, carbon type, radial distribution function (RDF), barysz matrix and molecular linear free energy relation. The suggested model could achieve satisfied square correlation coefficient of R2 = 0.72, higher than of many previous studies, indicating its superiority. Rigid validation criteria were met using external data with Q2 ˃ 0.5 and R2 = 0.75, reflecting the predictive power of the model. CONCLUSION: The QSAR model was applied for screening botanical components of herbal preparations used to treat BPH, and could predict the activity of some, among others, making reasonable attribution to the proposed effect of these preparations. Gamma tocopherol was found to be an active inhibitor, in consistence with many previous studies, anticipating the power of this model in the prediction of new candidate molecules and suggesting further investigations.

Modeling CNS Involvement in Pompe Disease Using Neural Stem Cells Generated from Patient-Derived Induced Pluripotent Stem Cells.

Pompe disease is a lysosomal storage disorder caused by autosomal recessive mutations in the acid alpha-glucosidase (GAA) gene. Acid alpha-glucosidase deficiency leads to abnormal glycogen accumulation in patient cells. Given the increasing evidence of central nervous system (CNS) involvement in classic infantile Pompe disease, we used neural stem cells, differentiated from patient induced pluripotent stem cells, to model the neuronal phenotype of Pompe disease. These Pompe neural stem cells exhibited disease-related phenotypes including glycogen accumulation, increased lysosomal staining, and secondary lipid buildup. These morphological phenotypes in patient neural stem cells provided a tool for drug efficacy evaluation. Two potential therapeutic agents, hydroxypropyl-ß-cyclodextrin and δ-tocopherol, were tested along with recombinant human acid alpha-glucosidase (rhGAA) in this cell-based Pompe model. Treatment with rhGAA reduced LysoTracker staining in Pompe neural stem cells, indicating reduced lysosome size. Additionally, treatment of diseased neural stem cells with the combination of hydroxypropyl-ß-cyclodextrin and δ-tocopherol significantly reduced the disease phenotypes. These results demonstrated patient-derived Pompe neural stem cells could be used as a model to study disease pathogenesis, to evaluate drug efficacy, and to screen compounds for drug discovery in the context of correcting CNS defects.

Comparative Effects of Alpha- and Gamma-Tocopherol on Mitochondrial Functions in Alzheimer's Disease In Vitro Model.

Vitamin E acts as an antioxidant and reduces the level of reactive oxygen species (ROS) in Alzheimer's disease (AD). Alpha-tocopherol (ATF) is the most widely studied form of vitamin E besides gamma-tocopherol (GTF) which also shows beneficial effects in AD. The levels of amyloid-beta (Aß) and amyloid precursor protein (APP) increased in the brains of AD patients, and mutations in the APP gene are known to enhance the production of Aß. Mitochondrial function was shown to be affected by the increased level of Aß and may induce cell death. Here, we aimed to compare the effects of ATF and GTF on their ability to reduce Aß level, modulate mitochondrial function and reduce the apoptosis marker in SH-SY5Y cells stably transfected with the wild-type or mutant form of the APP gene. The Aß level was measured by ELISA, the mitochondrial ROS and ATP level were quantified by fluorescence and luciferase assay respectively whereas the complex V enzyme activity was measured by spectrophotometry. The expressions of genes involved in the regulation of mitochondrial membrane permeability such as voltage dependent anion channel (VDAC1), adenine nucleotide translocase (ANT), and cyclophilin D (CYPD) were determined by quantitative real-time polymerase chain reaction (qRT-PCR), while the expressions of cyclophilin D (CypD), cytochrome c, Bcl2 associated X (BAX), B cell lymphoma-2 (Bcl-2), and pro-caspase-3 were determined by western blot. Our results showed that mitochondrial ROS level was elevated accompanied by decreased ATP level and complex V enzyme activity in SH-SY5Y cells expressing the mutant APP gene (p < 0.05). Treatment with both ATF and GTF reduced the mitochondrial ROS level with maximum reduction was observed in the cells treated with high concentrations of ATF and GTF (p < 0.05). However, only GTF at 80 µM significantly increase the ATP level and complex V enzyme activity (p < 0.05). VDAC1 and CYPD were downregulated and CypD protein was significantly overexpressed in cells transfected with the wild-type (WT) and mutant APP gene (p < 0.05). Cytochrome c release, the ratio of BAX/Bcl-2, and pro-caspase-3 expression increased in cells expressing mutated APP gene (p < 0.05). The expression of CypD and pro-caspase 3 protein, and the ratio of BAX/Bcl-2 were increased in the following order; SH-SY5Y-APP-WT < SH-SY5Y-APP Swe

Upregulation of pERK and c-JUN by γ-tocotrienol and not α-tocopherol are essential to the differential effect on apoptosis in prostate cancer cells.

BACKGROUND: α-tocopherol (AT) and γ-tocotrienol (GT3) are vitamin E isoforms considered to have potential chemopreventive properties. AT has been widely studied in vitro and in clinical trials with mixed results. The latest clinical study (SELECT trial) tested AT in prostate cancer patients, determined that AT provided no benefit, and could promote cancer. Conversely, GT3 has shown antineoplastic properties in several in vitro studies, with no clinical studies published to date. GT3 causes apoptosis via upregulation of the JNK pathway; however, inhibition results in a partial block of cell death. We compared side by side the mechanistic differences in these cells in response to AT and GT3. METHODS: The effects of GT3 and AT were studied on androgen sensitive LNCaP and androgen independent PC-3 prostate cancer cells. Their cytotoxic effects were analyzed via MTT and confirmed by metabolic assays measuring ATP. Cellular pathways were studied by immunoblot. Quantitative analysis and the determination of relationships between cell signaling events were analyzed for both agents tested. Non-cancerous prostate RWPE-1 cells were also included as a control. RESULTS: The RAF/RAS/ERK pathway was significantly activated by GT3 in LNCaP and PC-3 cells but not by AT. This activation is essential for the apoptotic affect by GT3 as demonstrated the complete inhibition of apoptosis by MEK1 inhibitor U0126. Phospho-c-JUN was upregulated by GT3 but not AT. No changes were observed on AKT for either agent, and no release of cytochrome c into the cytoplasm was detected. Caspases 9 and 3 were efficiently activated by GT3 on both cell lines irrespective of androgen sensitivity, but not in cells dosed with AT. Cell viability of non-cancerous RWPE-1 cells was affected neither by GT3 nor AT. CONCLUSIONS: c-JUN is a recognized master regulator of apoptosis as shown previously in prostate cancer. However, the mechanism of action of GT3 in these cells also include a significant activation of ERK which is essential for the apoptotic effect of GT3. The activation of both, ERK and c-JUN, is required for apoptosis and may suggest a relevant step in ensuring circumvention of mechanisms of resistance related to the constitutive activation of MEK1.

α-Tocopherol, but Not γ-Tocopherol, Attenuates the Expression of Selective Tumor Necrosis Factor-Alpha-Induced Genes in Primary Human Aortic Cell Lines.

Of the antioxidant vitamin E isoforms, α-tocopherol (αT) and γ-tocopherol (γT) are the most abundant in the human diet, and αT is consumed from both natural and synthetic sources. αT and γT may differentially impact inflammation and influence cardiovascular outcomes, in part by modulating gene expression. The goal of this study was to compare the effects of natural αT, synthetic αT, and γT on gene expression in two human cell lines. Human aortic smooth muscle cells (HASMC) and endothelial cells (HAEC) were either: (1) treated with 25 µM tocopherols alone, or (2) pretreated with tocopherols prior to a pro-inflammatory cytokine (tumor necrosis factor-alpha, TNF-α) stimulation. The expression of atherosclerosis-related genes was measured using RT2 Profiler PCR arrays. Tocopherol treatments alone did not significantly modulate the expression of genes in unstimulated HASMC or HAEC. TNF-α stimulation significantly upregulated genes involved with apoptosis and stress response in both cell lines. Pretreating cells with tocopherols did not normalize the gene expression changes induced by TNF-α. However, αT pretreatments, but not γT pretreatments, attenuated TNF expression in both HASMC and HAEC. These findings suggest that under stimulated conditions, αT modestly modulates the expression of selective genes and that αT may be more anti-inflammatory than γT.

δ-Tocopherol promotes thermogenic gene expression via PGC-1α upregulation in 3T3-L1 cells.

Activation of thermogenic adipocytes (brown and beige) has been considered an attractive target for weight loss and treatment of metabolic disease. Peroxisome proliferator-activated receptor γ co-activator-1 α (PGC1-α) is a master regulator of thermogenic gene expression in thermogenic adipocytes. We previously reported that α-tocopherol upregulated PGC-1α gene expression and promoted thermogenic adipocyte differentiation in mammalian adipocytes. In this study, we investigated the effects of the vitamin E analogs (α-, γ- and δ-tocopherol) on PGC-1α and uncoupling protein 1 (UCP1) gene expression in 3T3-L1 cells. The expression of PGC-1α and UCP1 increased significantly with the addition of δ-tocopherol. In δ-tocopherol-treated cells, nuclear translocation of PGC-1α increased, as did p38 mitogen-activated protein kinase (MAPK) expression and phosphorylation. Our results suggest that p38 MAPK activation by δ-tocopherol contributes to PGC-1α activation and UCP1 induction.

Gamma-tocotrienol attenuates the aberrant lipid mediator production in NLRP3 inflammasome-stimulated macrophages.

The activation of NLRP3 inflammasome in innate immune cells is associated with enhanced production of pro-inflammatory lipid mediator eicosanoids that play a crucial role in propagating inflammation. Gamma-tocotrienol (γT3) is an unsaturated vitamin E that has been demonstrated to attenuate NLRP3-inflammasome. However, the role of γT3 in regulating eicosanoid formation is unknown. We hypothesized that γT3 abolishes the eicosanoid production by modulating the macrophage lipidome. LPS-primed bone marrow-derived macrophages (BMDM) were stimulated with saturated fatty acids (SFA) along with γT3, and the effects of γT3 in modulating macrophage lipidome were quantified by using mass spectrometry based-shotgun lipidomic approaches. The SFA-mediated inflammasome activation induced robust changes in lipid species of glycerolipids (GL), glycerophospholipids (GPL), and sphingolipids in BMDM, which were distinctly different in the γT3-treated BMDM. The γT3 treatment caused substantial decreases of lysophospholipids (LysoPL), diacylglycerol (DAG), and free arachidonic acid (AA, C20:4), indicating that γT3 limits the availability of AA, the precursor for eicosanoids. This was confirmed by the pulse-chase experiment using [3H]-AA, and by diminished prostaglandin E2 (PGE2) secretion by ELISA. Concurrently, γT3 inhibited LPS-induced cyclooxygenases 2 (COX2) induction, further suppressing prostaglandin synthesis. In addition, γT3 attenuated ceramide synthesis by transcriptional downregulation of key enzymes for de novo synthesis. The altered lipid metabolism during inflammation is linked to reduced ATP production, which was partly rescued by γT3. Taken together, our work revealed that γT3 induces distinct modification of the macrophage lipidome to reduce AA release and corresponding lipid mediator synthesis, leading to attenuated cellular lipotoxicity.

Role of dietary α- and γ-tocopherol from Rosa mosqueta oil in the prevention of alterations induced by high-fat diet in a murine model.

OBJECTIVE: The aim of this study was to evaluate the contribution of tocopherols present in Rosa mosqueta oil (RM) in the prevention of high-fat diet (HFD)-induced alterations. METHODS: Male C57 BL/6 J mice (n = 9/group) were fed for 12 wk and divided into four groups: control (CD; 10% kcal fat, 20% kcal protein, 70% kcal carbohydrates); HFD (60% as fat, 20% kcal protein, 20% kcal carbohydrates); HFD + RM (0.01 mL/g body weight/d); and HFD + RM- without tocopherols (0.01 mL/g body weight/d). Parameters of obesity, liver steatosis (histology, triacylglycerols content), inflammation (adipose NLRP3 inflammasome, tumor necrosis factor-α and interleukin-1 ß expression, hepatic nuclear factor-κB) and oxidative stress (hepatic Nrf2 activation, carbonylated proteins) were evaluated. RESULTS: Liver steatosis, inflammatory, and oxidative stress parameters were significantly (P < 0.05) increased in the HFD + RM- compared with the HFD + RM, with no differences between HFD and HFD + RM-. CONCLUSION: The present study suggests that α- and γ-tocopherols from RM may have an important role in the prevention of alterations induced by HFD.

Gamma tocopherol-enriched supplement reduces sputum eosinophilia and endotoxin-induced sputum neutrophilia in volunteers with asthma.

BACKGROUND: We and others have shown that the gamma tocopherol (γT) isoform of vitamin E has multiple anti-inflammatory and antioxidant actions and that γT supplementation reduces eosinophilic and endotoxin (LPS)-induced neutrophilic airway inflammation in animal models and healthy human volunteers. OBJECTIVE: We sought to determine whether γT supplementation reduces eosinophilic airway inflammation and acute neutrophilic response to inhaled LPS challenge in volunteers with asthma. METHODS: Participants with mild asthma were enrolled in a double-blinded, placebo-controlled crossover study to assess the effect of 1200 mg of γT daily for 14 days on sputum eosinophils, mucins, and cytokines. We also assessed the effect on acute inflammatory response to inhaled LPS challenge following γT treatment, focusing on changes in sputum neutrophilia, mucins, and cytokines. Mucociliary clearance was measured using gamma scintigraphy. RESULTS: Fifteen subjects with mild asthma completed both arms of the study. Compared with placebo, γT notably reduced pre-LPS challenge sputum eosinophils and mucins, including mucin 5AC and reduced LPS-induced airway neutrophil recruitment 6 and 24 hours after challenge. Mucociliary clearance was slowed 4 hours postchallenge in the placebo group but not in the γT treatment group. Total sputum mucins (but not mucin 5AC) were reduced at 24 hours postchallenge during γT treatment compared with placebo. CONCLUSIONS: When compared with placebo, γT supplementation for 14 days reduced inflammatory features of asthma, including sputum eosinophils and mucins, as well as acute airway response to inhaled LPS challenge. Larger scale clinical trials are needed to assess the efficacy of γT supplements as a complementary or steroid-sparing treatment for asthma.

Natural Forms of Vitamin E as Effective Agents for Cancer Prevention and Therapy.

Initial research on vitamin E and cancer has focused on α-tocopherol (αT), but recent clinical studies on cancer-preventive effects of αT supplementation have shown disappointing results, which has led to doubts about the role of vitamin E, including different vitamin E forms, in cancer prevention. However, accumulating mechanistic and preclinical animal studies show that other forms of vitamin E, such as γ-tocopherol (γT), δ-tocopherol (δT), γ-tocotrienol (γTE), and δ-tocotrienol (δTE), have far superior cancer-preventive activities than does αT. These vitamin E forms are much stronger than αT in inhibiting multiple cancer-promoting pathways, including cyclo-oxygenase (COX)- and 5-lipoxygenase (5-LOX)-catalyzed eicosanoids, and transcription factors such as nuclear transcription factor κB (NF-κB) and signal transducer and activator of transcription factor 3 (STAT3). These vitamin E forms, but not αT, cause pro-death or antiproliferation effects in cancer cells via modulating various signaling pathways, including sphingolipid metabolism. Unlike αT, these vitamin E forms are quickly metabolized to various carboxychromanols including 13'-carboxychromanols, which have even stronger anti-inflammatory and anticancer effects than some vitamin precursors. Consistent with mechanistic findings, γT, δT, γTE, and δTE, but not αT, have been shown to be effective for preventing the progression of various types of cancer in preclinical animal models. This review focuses on cancer-preventive effects and mechanisms of γT, δT, γTE, and δTE in cells and preclinical models and discusses current progress in clinical trials. The existing evidence strongly indicates that these lesser-known vitamin E forms are effective agents for cancer prevention or as adjuvants for improving prevention, therapy, and control of cancer.

Differential Gene Regulation and Tumor-Inhibitory Activities of Alpha-, Delta-, and Gamma-Tocopherols in Estrogen-Mediated Mammary Carcinogenesis.

Despite experimental evidence elucidating the antitumor activities of tocopherols, clinical trials with α-tocopherol (α-T) have failed to demonstrate its beneficial effects in cancer prevention. This study compared the chemopreventive efficacy of individual tocopherols (α-, δ-, and γ-T) and a γ-T-rich tocopherol mixture (γ-TmT) in the August-Copenhagen Irish (ACI) rat model of estrogen-mediated mammary cancer. Female ACI rats receiving 17ß-estradiol (E2) implants were administered with 0.2% α-T, δ-T, γ-T, or γ-TmT for 30 weeks. Although α-T had no significant effects on mammary tumor growth in ACI rats, δ-T, γ-T, and γ-TmT reduced mammary tumor volume by 51% (P < 0.05), 60% (P < 0.01), and 59% (P < 0.01), respectively. Immunohistochemical analysis revealed that δ-T, γ-T, and γ-TmT reduced levels of the cell proliferation marker, proliferating cell nuclear antigen, in the rat mammary tumors. To gain further insight into the biological functions of different forms of tocopherols, RNA-seq analysis of the tumors was performed. Treatment with γ-T induced robust gene expression changes in the mammary tumors of ACI rats. Ingenuity Pathway Analysis identified "Cancer" as a top disease pathway and "Tumor growth" and "Metastasis" as the top signaling pathways modulated by γ-T. Although the results need further functional validation, this study presents an unbiased attempt to understand the differences between biological activities of individual forms of tocopherols at the whole transcriptome level. In conclusion, δ-T and γ-T have superior cancer preventive properties compared to α-T in the prevention of estrogen-mediated mammary carcinogenesis. Cancer Prev Res; 10(12); 694-703. ©2017 AACR.

γ-Tocotrienol-induced disruption of lipid rafts in human breast cancer cells is associated with a reduction in exosome heregulin content.

Overexpression of heregulin, a potent ligand that activates HER3 and HER4 receptors, plays a significant role in the development of chemotherapy resistance in breast cancer patients. Exosomes released from cancer cells are small vesicles originating from the outward budding of lipid rafts that carry various mitogenic proteins that then act locally in an autocrine/paracrine manner to stimulate cancer cell growth. Since the anticancer activity of γ-tocotrienol has been shown to be mediated in part through the disruption of lipid rafts, studies were conducted to determine the effect of γ-tocotrienol on exosomes mitogenic biopotency. Exosomes isolated from the media of cultured T47D breast cancer cells were found to stimulate T47D cell growth in a dose-dependent manner. These growth stimulating effects were due to the high levels of heregulin contained in the exosomes that act to stimulate HER3 and HER4 activation, heterodimerization and mitogenic signaling. Exposure to 5 µM γ-tocotrienol resulted in the selective accumulation and disruption in the integrity of the lipid raft microdomain and a corresponding decrease in exosome heregulin content and mitogenic biopotency. These findings provide strong evidence indicating that the anticancer effects of γ-tocotrienol are mediated, at least in part, by directly disrupting HER dimerization and signaling within the lipid rafts and indirectly by reducing exosome heregulin content and subsequent autocrine/paracrine mitogenic stimulation.