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Salinomycin and its derivatives display promising anti-proliferating activity against bloodstream forms of Trypanosoma brucei . The mechanism of trypanocidal action of these compounds is due to their ionophoretic activity inducing an influx of sodium cations followed by osmotic water uptake, leading to massive swelling of bloodstream-form trypanosomes. Generally, higher trypanocidal activities of salinomycin derivatives are associated with higher cell swelling activities. Although ironomycin (C20-propargylamine derivative of salinomycin) and salinomycin showed identical cell swelling activities, ironomycin was 6 times more trypanocidal than salinomycin, and the 50% growth inhibition (GI 50 ) values were 0.034 μM and 0.20 μM, respectively. However, when bloodstream-form trypanosomes were incubated with ironomycin in the presence of vitamin E and ammonium ferric citrate, the trypanocidal activity of the compound was reduced to that of salinomycin (GI 50 = 0.21 μM vs. GI 50 = 0.20 μM). In addition, vitamin E was found to decrease the trypanocidal activity of ironomycin much more than ammonium ferric citrate (GI 50 = 0.18 μM vs. GI 50 = 0.042 μM). Moreover, ironomycin caused a reduction in the uptake of the iron-carrier protein transferrin mediated by a downregulation of the transferrin receptor and led to the accumulation and sequestering of iron(II) in the parasite's lysosome, triggering an increase production of reactive oxygen species (ROS). These results suggest that the increased trypanocidal activity of ironomycin can be mainly attributed to an increased ROS production and, to a lesser extent, an impairment in iron uptake.

Cancer is a disease resulting from uncontrolled cell division, which significantly contributes to human mortality rates. An alternative approach to cancer treatment, such as cancer immunotherapy, is needed as the existing chemotherapy and radiotherapy approaches target the cancer cells and healthy dividing cells. Vitamin E is a plant-derived lipid-soluble antioxidant with numerous health-promoting benefits, including anticancer and immunomodulatory properties. Vitamin E comprises eight natural isoforms: tocopherols ( α , β , δ , and γ ) and tocotrienols ( α , β , δ , and γ ). While initial research focused on the anticancer properties of α -tocopherol, there is growing interest in other natural forms and modified synthetic analogues of vitamin E due to their unique properties and enhanced anticancer effects. Hence, this review is aimed at outlining the effect of vitamin E and its analogues at various steps of the cancer-immunity cycle that can be used to stimulate anticancer immune responses., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Nevvin Raaj Morgan et al.)

In the pathological process of Alzheimer's disease, neuronal cell death is closely related to the accumulation of reactive oxygen species. Our previous studies have found that oxidative stress can activate microtubule affinity-regulating kinases, resulting in elevated phosphorylation levels of tau protein specifically at the Ser262 residue in N1E-115 cells that have been subjected to exposure to hydrogen peroxide. This process may be one of the pathogenic mechanisms of Alzheimer's disease. Vitamin E is a fat-soluble, naturally occurring antioxidant that plays a crucial role in biological systems. This study aimed to examine the probable processes that contribute to the inhibiting effect on the abnormal phosphorylation of tau protein and the neuroprotective activity of a particular type of vitamin E, α-tocotrienol. The experimental analysis revealed that α-tocotrienol showed significant neuroprotective effects in the N1E-115 cell line. Our data further suggest that one of the mechanisms underlying the neuroprotective effects of α-tocotrienol may be through the inhibition of microtubule affinity-regulated kinase activation, which significantly reduces the oxidative stress-induced aberrant elevation of p-Tau (Ser262) levels. These results indicate that α-tocotrienol may represent an intriguing strategy for treating or preventing Alzheimer's disease.

Exposure to high, marginally lethal doses or higher of ionizing radiation, either intentional or accidental, results in injury to various organs. Currently, there is only a limited number of safe and effective radiation countermeasures approved by US Food and Drug Administration for such injuries. These approved agents are effective for only the hematopoietic component of the acute radiation syndrome and must be administered only after the exposure event: currently, there is no FDA-approved agent that can be used prophylactically. The nutraceutical, gamma-tocotrienol (GT3) has been found to be a promising radioprotector of such exposure-related injuries, especially those of a hematopoietic nature, when tested in either rodents or nonhuman primates. We investigated the nature of injuries and the possible protective effects of GT3 within select organ systems/tissues caused by both non-lethal level (4.0 Gy), as well as potentially lethal level (5.8 Gy) of ionizing radiation, delivered as total-body or partial-body exposure. Results indicated that the most severe, dose-dependent injuries occurred within those organ systems with strong self-renewing capacities (e.g., the lymphohematopoietic and gastrointestinal systems), while in other tissues (e.g., liver, kidney, lung) endowed with less self-renewal, the pathologies noted tended to be less pronounced and less dependent on the level of exposure dose or on the applied exposure regimen. The prophylactic use of the test nutraceutical, GT3, appeared to limit the extent of irradiation-associated pathology within blood forming tissues and, to some extent, within the small intestine of the gastrointestinal tract. No distinct, global pattern of bodily protection was noted with the agent's use, although a hint of a possible radioprotective benefit was suggested not only by a lessening of apparent injury within select organ systems, but also by way of noting the lack of early onset of moribundity within select GT3-treated animals., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Despite remarkable scientific progress over the past six decades within the medical arts and in radiobiology in general, limited radiation medical countermeasures (MCMs) have been approved by the United States Food and Drug Administration for the acute radiation syndrome (ARS). Additional effort is needed to develop large animal models for improving the prediction of clinical safety and effectiveness of MCMs for acute and delayed effects of radiation in humans. Nonhuman primates (NHPs) are considered the animal models that reproduce the most appropriate representation of human disease and are considered the gold standard for drug development and regulatory approval. The clinical and histopathological effects of supralethal, total- or partial-body irradiations (12 Gy) of NHPs were assessed, along with possible protective actions of a promising radiation MCM, gamma-tocotrienol (GT3). Results show that these supralethal radiation exposures induce severe injuries that manifest both clinically as well as pathologically, as evidenced by the noted functionally crippling lesions within various major organ systems of experimental NHPs. The MCM, GT3, has limited radioprotective efficacy against such supralethal radiation doses., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Objective: We aimed to compare the efficacy of δ-tocotrienol with α-tocopherol in the treatment of patients with non-alcoholic fatty liver disease (NAFLD)., Design and Interventions: This study was a double-blinded, active-controlled trial. The patients with NAFLD were randomly assigned to receive either δ-tocotrienol 300 mg or α-tocopherol 268 mg twice daily for 48 weeks., Endpoints: The primary endpoints were change from baseline in fatty liver index (FLI), liver-to-spleen attenuation ratio (L/S ratio), and homeostatic model assessment for insulin resistance (HOMA-IR) at 48 weeks. Key secondary endpoints were change in markers of inflammation, oxidative stress, and hepatocyte apoptosis. Clinical assessment, biochemical analysis, and computed tomography scan of the liver were conducted at baseline, 24 and 48 weeks., Results: A total of 100 patients (δ-tocotrienol = 50, α-tocopherol = 50) were randomized and included in the intention to treat analysis. Compared with baseline, there was a significant improvement (p < .001) in FLI, L/S ratio, HOMA-IR, and serum malondialdehyde in both groups at 48 weeks that was not significant between the two groups. However, there was a significantly greater decrease in body weight, serum interleukin-6, tumor necrosis factor-alpha, leptin, cytokeratin-18, and increase in adiponectin in the δ-tocotrienol group compared to the α-tocopherol group at 48 weeks (p < .05). No adverse events were reported., Conclusion: δ-tocotrienol and α-tocopherol exerted equally beneficial effects in terms of improvement in hepatic steatosis, oxidative stress, and insulin resistance in patients with NAFLD. However, δ-tocotrienol was more potent than α-tocopherol in reducing body weight, inflammation, and apoptosis associated with NAFLD. TRIAL REGISTRATION: Sri Lankan Clinical Trials Registry (https://slctr.lk/SLCTR/2019/038)., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Warburg effect is commonly recognized as a hallmark of nearly all tumors. In prostate cancer (PCa), it has been shown to be driven by PTEN loss- and Akt hyperactivation-associated upregulation of hexokinase 2 (HK2). δ-Tocotrienol (δ-TT) is an extensively studied antitumor compound; however, its role in affecting PCa glycolysis is still unclear. Herein, we demonstrated that δ-TT inhibits glucose uptake and lactate production in PTEN-deficient LNCaP and PC3 PCa cells, by specifically decreasing HK2 expression. Notably, this was accompanied by the inhibition of the Akt pathway. Moreover, the nutraceutical could synergize with the well-known hypoglycemic agent metformin in inducing PCa cell death, highlighting the crucial role of the above metabolic phenotype in δ-TT-mediated cytotoxicity. Collectively, these results unravel novel inhibitory effects of δ-TT on glycolytic reprogramming in PCa, thus providing new perspectives into the mechanisms of its antitumor activity and into its use in combination therapy.

The gastrointestinal (GI) system is highly susceptible to irradiation. Currently, there is no Food and Drug Administration (FDA)-approved medical countermeasures for GI radiation injury. The vitamin E analog gamma-tocotrienol (GT3) is a promising radioprotector in mice and nonhuman primates (NHP). We evaluated GT3-mediated GI recovery in total-body irradiated (TBI) NHPs. Sixteen rhesus macaques were divided into two groups; eight received vehicle and eight GT3 24 h prior to 12 Gy TBI. Proximal jejunum was assessed for structural injuries and crypt survival on day 4 and 7. Apoptotic cell death and crypt cell proliferation were assessed with TUNEL and Ki-67 immunostaining. Irradiation induced significant shortening of the villi and reduced mucosal surface area. GT3 induced an increase in crypt depth at day 7, suggesting that more stem cells survived and proliferated after irradiation. GT3 did not influence crypt survival after irradiation. GT3 treatment caused a significant decline in TUNEL-positive cells at both day 4 (p < 0.03) and 7 (p < 0.0003). Importantly, GT3 induced a significant increase in Ki-67-positive cells at day 7 (p < 0.05). These data suggest that GT3 has radioprotective function in intestinal epithelial and crypt cells. GT3 should be further explored as a prophylactic medical countermeasure for radiation-induced GI injury.

Vitamin E compounds, consisting of α, β, γ, and δ forms of tocopherols and tocotrienols, display different cancer preventive activities in experimental models. Tocotrienols may have higher potential for clinical use due to their lower effective doses in laboratory studies. However, most studies on tocotrienols have been carried out using cancer cell lines. Strong data from animal studies may encourage the use of tocotrienols for human cancer prevention research. To examine the cancer inhibitory activity of different vitamin E forms, we first investigated their inhibitory activities of different vitamin E forms in prostate cancer cell lines. We found that δ-tocotrienol (δT3) was the most effective form in inhibiting cell growth at equivalent doses. Because of this in vitro potency, δT3 was further studied using prostate-specific Pten-/- (Ptenp-/-) mice. We found that 0.05% δT3 in diet reduced prostate adenocarcinoma multiplicity by 32.7%, featuring increased apoptosis and reduced cell proliferation. The inhibitory effect of 0.05% δT3 in diet was similar to that of 0.2% δ-tocopherol (δT) in diet reported previously. Our further study on the δT3-induced transcriptome changes indicated that δT3 inhibited genes in blood vessel development in the prostate of Ptenp-/- mice, which was confirmed by IHC. Together, our results demonstrate that δT3 effectively inhibits the development of prostate adenocarcinoma in Ptenp-/- mice, which involves inhibition of proliferation and angiogenesis and promotion of apoptosis., Prevention Relevance: We demonstrated that δ-tocotrienol is the most active vitamin E form in inhibiting the growth of several prostate cancer cell lines. In transgenic Ptenp-/- mice, δ-tocotrienol inhibited the formation of prostate cancer. This result would encourage and help design clinical studies for the application of δ-tocotrienol for prostate cancer prevention., (©2022 American Association for Cancer Research.)

Radiological incidents or terrorist attacks would likely expose civilians and military personnel to high doses of ionizing radiation, leading to the development of acute radiation syndrome. We examined the effectiveness of prophylactic administration of a developmental radiation countermeasure, γ-tocotrienol (GT3), in a total-body irradiation (TBI) mouse model. CD2F1 mice received GT3 24 h prior to 11 Gy cobalt-60 gamma-irradiation. This dose of radiation induces severe hematopoietic acute radiation syndrome and moderate gastrointestinal injury. GT3 provided 100% protection, while the vehicle control group had 100% mortality. Two-dimensional differential in-gel electrophoresis was followed by mass spectrometry and Ingenuity Pathway Analysis (IPA). Analysis revealed a change in expression of 18 proteins in response to TBI, and these changes were reversed with prophylactic treatment of GT3. IPA revealed a network of associated proteins involved in cellular movement, immune cell trafficking, and inflammatory response. Of particular interest, significant expression changes in beta-2-glycoprotein 1, alpha-1-acid glycoprotein 1, alpha-2-macroglobulin, complement C3, mannose-binding protein C, and major urinary protein 6 were noted after TBI and reversed with GT3 treatment. This study reports the untargeted approach, the network, and specific serum proteins which could be translated as biomarkers of both radiation injury and protection by countermeasures., (© 2022. The Author(s).)

Objective: To study the effects of delta-tocotrienol on glycaemic control parameters in individuals with pre-diabetes., Methods: The randomised control trial was conducted at the Armed Forces Institute of Pathology, Rawalpindi, Pakistan, from July 15 to November 15, 2019, and comprised individuals aged 18-60 years having fasting plasma glucose of 5.6 to 6.9 mmol/L or glycosylated haemoglobin of 5.7 to 6.4%. They were randomised into group A receiving 300mg delta-tocotrienol and group B receiving a placebo once daily for 12 weeks. Weight, height, waist circumference, fasting plasma glucose, insulin and glycosylated haemoglobin were measured at the beginning and end of the trial to assess any change. Body mass index and homeostatic model assessment-insulin resistance were also calculated. Data was analysed using SPSS 21., Results: Of the 77participants, 40(52%) were in group A and 37(48%) in group B. Group A showed significantly greater reduction in terms of fasting plasma glucose, glycosylated haemoglobin, insulin and homeostatic model assessment-insulin resistance index (p≤0.001) post-intervention., Conclusions: Delta-tocotrienol supplementation was found to have a significant effect in improving glycaemic control parameters in persons with pre-diabetes. Futures larger scale clinical trials are needed to confirm these findings., Clinical Trial Number: SLCTR/2019/024.

Preeclampsia (PE) is a severe medical disorder during pregnancy and there has been controversy about the effects of vitamin E on PE. This research intended to explore if δ-tocotrienol (δ-TT), an isomer of vitamin E, could impact PE. Preeclamptic and normal placentas were obtained and total RNA was extracted. The expression of different genes was analyzed through quantitative real-time polymerase chain reaction (qRT-PCR) and Pearson correlation analysis was conducted. After that, HTR-8/SVneo cells (human trophoblasts) were chosen and they were subjected to δ-tocotrienol treatment and then Cell Counting Kit-8 was used to test cell viability. To assess the effects of δ-TT on trophoblasts, wound healing assay and Transwell invasion assay were performed. How miR-429 interacts with ZEB1 was examined via dual luciferase reporter assay. Also, protein expression was evaluated via Western blotting. Our results have shown that δ-TT can impair the viability of trophoblasts and induce their apoptosis. Additionally, it can repress the growth, migration, epithelial-mesenchymal transition (EMT), invasion and angiogenesis in trophoblasts. Mechanistically, δ-TT exerts these effects on trophoblasts via downregulating miR-429 and upregulating ZEB1. Furthermore, miR-429 can bind ZEB1 directly. Clinical sample analysis has revealed that miR-429 expression in preeclamptic placenta is higher than that in normal placenta, but ZEB1 expression in preeclamptic placenta is downregulated. Also, there is a negative association between miR-429 and ZEB1 expression in preeclamptic placentas. These discoveries imply that δ-TT may be hazardous to pregnancy and should not be used in preeclamptic patients. In addition, targeting miR-429 might treat PE.

Context: Glucagon-like peptide 1 (GLP-1) and α-tocopheryl quinone can promote the growth of intestinal flora and affect the pathogenesis of non-alcoholic steatohepatitis (NASH)., Objective: This study determines the molecular mechanism of the effect of tocopheryl quinone in the treatment of high cholesterol and cholate diet (HFCC)-induced NASH., Materials and Methods: Thirty-two male Sprague Dawley (SD) rats grouped as lean control (LC), LC + tocopheryl quinone (1 mL of 3 × 10 6 dpm tocopheryl quinone via i.p. injection), HFCC (5.1 kcal/g of fat diet), and HFCC + tocopheryl quinone. Profiles of intestinal flora were assessed by 16S ribosomal ribonucleic acid-based analysis. Levels and activity of GLP-1, interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) in intestinal tissues were detected by immunohistochemistry (IHC), Western blot and enzyme-linked immunosorbent assay (ELISA)., Results: HFCC rats presented higher levels of cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL), while tocopheryl quinone reversed the effects of HFCC. HFCC dysregulated malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), Vitamin E, 12-hydroxyeicosatetraenoic acid (12-HETE), 13-hydroxyoctadecadienoic acid (13-HODE) and nuclear factor kappa B (NF-κB), and the effects of HFCC were reversed by the treatment of tocopheryl quinone. Also, GLP-1 in the HFCC group was down-regulated while the IL-6 and TNF-α activity and endotoxins were all up-regulated. HFCC significantly decreased the number and diversity of bacteria, whereas tocopheryl quinone substantially restored the balance of intestinal flora and promoted the growth of both Bacteroides and Lactobacilli in vitro ., Discussion and Conclusions: α-Tocopheryl quinone relieves HFCC-induced NASH via regulating oxidative stress, GLP-1 expression, intestinal flora imbalance, and the metabolism of glucose and lipids.

Ischemia-induced mitochondrial dysfunction and ATP depletion in the kidney result in disruption of primary functions and acute injury of the kidney. This study tested whether γ-tocotrienol (GTT), a member of the vitamin E family, protects mitochondrial function, reduces ATP deficits, and improves renal functions and survival after ischemia/reperfusion injury. Vehicle or GTT (200 mg/kg) were administered to mice 12 h before bilateral kidney ischemia, and endpoints were assessed at different timepoints of reperfusion. GTT treatment reduced decreases in state 3 respiration and accelerated recovery of this function after ischemia. GTT prevented decreases in activities of complexes I and III of the respiratory chain, and blocked ischemia-induced decreases in F 0 F 1 -ATPase activity and ATP content in renal cortical tissue. GTT improved renal morphology at 72 h after ischemia, reduced numbers of necrotic proximal tubular and inflammatory cells, and enhanced tubular regeneration. GTT treatment ameliorated increases in plasma creatinine levels and accelerated recovery of creatinine levels after ischemia. Lastly, 89% of mice receiving GTT and 70% of those receiving vehicle survived ischemia. Conclusions: Our data show novel observations that GTT administration improves mitochondrial respiration, prevents ATP deficits, promotes tubular regeneration, ameliorates decreases in renal functions, and increases survival after acute kidney injury in mice.

Objectives: Among gynaecologic malignancies, ovarian cancer (OC) represents the leading cause of death for women worldwide. Current OC treatment involves cytoreductive surgery followed by platinum-based chemotherapy, which is associated with severe side effects and development of drug resistance. Therefore, new therapeutic strategies are urgently needed. Herein, we evaluated the anti-tumour effects of Vitamin E-derived δ-tocotrienol (δ-TT) in two human OC cell lines, IGROV-1 and SKOV-3 cells., Materials and Methods: MTT and Trypan blue exclusion assays were used to assess δ-TT cytotoxicity, alone or in combination with other molecules. δ-TT effects on cell cycle, apoptosis, ROS generation and MAPK phosphorylation were investigated by flow cytometry, Western blot and immunofluorescence analyses. The synergism between δ-TT and chemotherapy was evaluated by isobologram analysis., Results: We demonstrated that δ-TT could induce cell cycle block at G1-S phase and mitochondrial apoptosis in OC cell lines. In particular, we found that the proapoptotic activity of δ-TT correlated with mitochondrial ROS production and subsequent JNK and p38 activation. Finally, we observed that the compound was able to synergize with cisplatin, not only enhancing its cytotoxicity in IGROV-1 and SKOV-3 cells but also re-sensitizing IGROV-1/Pt1 cell line to its anti-tumour effects., Conclusions: δ-TT triggers G1 phase cell cycle arrest and ROS/MAPK-mediated apoptosis in OC cells and sensitizes them to platinum treatment, thus representing an interesting option for novel chemopreventive/therapeutic strategies for OC., (© 2021 The Authors. Cell Proliferation published by John Wiley & Sons Ltd.)

Nutrition can modulate host immune responses as well as promote anticancer effects. In this study, two nutritional supplements, namely gamma-tocotrienol (γT3) and Spirulina, were evaluated for their immune-enhancing and anticancer effects in a syngeneic mouse model of breast cancer (BC). Five-week-old female BALB/c mice were fed Spirulina , γT3, or a combination of Spirulina and γT3 ( Spirulina + γT3) for 56 days. The mice were inoculated with 4T1 cells into their mammary fat pad on day 28 to induce BC. The animals were culled on day 56 for various analyses. A significant reduction ( p < 0.05) in tumor volume was only observed on day 37 and 49 in animals fed with the combination of γT3 + Spirulina . There was a marked increase ( p < 0.05) of CD4/CD127 + T-cells and decrease ( p < 0.05) of T-regulatory cells in peripheral blood from mice fed with either γT3 or Spirulina. The breast tissue of the combined group showed abundant areas of necrosis, but did not prevent metastasis to the liver. Although there was a significant increase ( p < 0.05) of MIG-6 and Cadherin 13 expression in tumors from γT3-fed animals, there were no significant ( p > 0.05) differences in the expression of MIG-6, Cadherin 13, BIRC5, and Serpine1 upon combined feeding. This showed that combined γT3 + Spirulina treatment did not show any synergistic anticancer effects in this study model.

With the successful development and increased use of targeted radionuclide therapy for treating cancer comes the increased risk of radiation injury to bone marrow-both direct suppression and stochastic effects, leading to neoplasia. Herein, we report a novel radioprotector drug, a liposomal formulation of γ-tocotrienol (GT3), or GT3-Nano for short, to mitigate bone marrow radiation damage during targeted radionuclide therapy. Methods: GT3 was loaded into liposomes using passive loading. 64 Cu-GT3-Nano and 3 H-GT3-Nano were synthesized to study the in vivo biodistribution profile of the liposome and GT3 individually. The radioprotection efficacy of GT3-Nano was assessed after acute 137 Cs whole-body irradiation at a sublethal (4 Gy), a lethal (9 Gy), or a single high-dose administration of 153 Sm-ethylenediamine- N,N,N',N' -tetrakis(methylene phosphonic acid) (EDTMP). Flow cytometry and fluorescence microscopy were used to analyze hematopoietic cell population dynamics and the cellular site of GT3-Nano localization in the spleen and bone marrow, respectively. Results: Bone marrow uptake and retention (percentage injected dose per gram of tissue) at 24 h was 6.98 ± 2.34 for 64 Cu-GT3-Nano and 7.44 ± 2.52 for 3 H-GT3-Nano. GT3-Nano administered 24 h before or after 4 Gy of total-body irradiation (TBI) promoted rapid and complete hematopoietic recovery, whereas recovery of controls stalled at 60%. GT3-Nano demonstrated dose-dependent radioprotection, achieving 90% survival at 50 mg/kg against lethal 9-Gy TBI. Flow cytometry of the bone marrow indicated that progenitor bone marrow cells MPP2 and CMP were upregulated in GT3-Nano-treated mice. Immunohistochemistry showed that GT3-Nano accumulates in CD105-positive sinusoid epithelial cells. Conclusion: GT3-Nano is highly effective in mitigating the marrow-suppressive effects of sublethal and lethal TBI in mice. GT3-Nano can facilitate rapid recovery of hematopoietic components in mice treated with the endoradiotherapeutic agent 153 Sm-EDTMP., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

This study aimed to compare the distribution of vitamin E analogs, particularly α-tocopherol and δ-tocopherol, in mice fed with a normal diet and a high-fat and high-sucrose diet separately. We used male C57BL/6JJcl strain mice, which were divided into six groups (control [C], Cα, Cδ, high-fat and high-sucrose [H], Hα, and Hδ groups) and bred for 4 weeks. The additional quantity of α-tocopherol or E-mix D (containing 86.7% δ-tocopherol) into diet was 800 mg/kg diet. The final body weight was significantly higher in the H group than in the C group. However, the effects of vitamin E analog intake had no significant difference, with no synergy between vitamin E and diet. Similar results were obtained in epididymal fat weight. Moreover, α-tocopherol was mainly distributed in the liver in both the Cα group and Hα group, whereas δ-tocopherol mostly accumulated in the epididymal fat, in both the Cδ group and Hδ group. Also, δ-tocopherol was detected in all tissues in both groups. Both the α-tocopherol and δ-tocopherol levels in the epididymal fat were significantly lower in the H group than in the C group. In conclusion, our results suggest that a portion of δ-tocopherol was incorporated into the adipose tissue by chylomicron before arriving at the liver, and then it is metabolized in the liver.

Garcinoic acid has been identified as an inhibitor of DNA polymerase β (pol β). However, no structure-activity relationship (SAR) studies of garcinoic acid as a pol β inhibitor have been conducted, in part due to the lack of an efficient synthetic method for this natural product and its analogs. We developed an efficient semi-synthetic method for garcinoic acid and its analogs by starting from natural product δ-tocotrienol. Our preliminary SAR studies provided a valuable insight into future discovery of garcinoic acid-based pol β inhibitors.

Chronic low-grade inflammation is a primary characteristic of obesity and can lead to other metabolic complications including insulin resistance and type 2 diabetes (T2D). Several anti-inflammatory dietary bioactives decrease inflammation that accompanies metabolic diseases. We are specifically interested in delta-tocotrienol, (DT3) an isomer of vitamin E, and tart cherry anthocyanins (TCA), both of which possess individual anti-inflammatory properties. We have previously demonstrated that DT3 and TCA, individually, reduced systemic and adipose tissue inflammation in rodent models of obesity. However, whether these compounds have combinatorial effects has not been determined yet. Hence, we hypothesize that a combined treatment of DT3 and TCA will have great effects in reducing inflammation in adipocytes, and that these effects are mediated via the nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), a major inflammatory transcription factor. We used 3T3-L1 adipocytes and treated them with 1-5 µM doses of DT3 along with tart cherry containing 18-36 µg anthocyanin/mL, to assess effects on inflammation. Neither DT3 nor TCA, nor their combinations had toxic effects on adipocytes. Furthermore, pro-inflammatory markers interleukin-6 (IL-6) and p-65 (subunit of NFkB) were reduced at the protein level in media collected from adipocytes with both individual and combined treatments. Additionally, other downstream targets of NFkB including macrophage inflammatory protein 2 ( Mip2 ), and Cyclooxygenase-2 ( Cox2 ) were also significantly downregulated ( p ≤ 0.05) when treated with individual and combined doses of DT3 and TCA with no additional combinatorial effects. In summary, DT3 and TCA individually, are beneficial in reducing inflammation with no additional combinatorial effects.

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Garcinoic acid (GA or δ-T3-13'COOH), is a natural vitamin E metabolite that has preliminarily been identified as a modulator of nuclear receptors involved in β-amyloid (Aβ) metabolism and progression of Alzheimer's disease (AD). In this study, we investigated GA's effects on Aβ oligomer formation and deposition. Specifically, we compared them with those of other vitamin E analogs and the soy isoflavone genistein, a natural agonist of peroxisome proliferator-activated receptor γ (PPARγ) that has therapeutic potential for managing AD. GA significantly reduced Aβ aggregation and accumulation in mouse cortical astrocytes. Similarly to genistein, GA up-regulated PPARγ expression and apolipoprotein E (ApoE) efflux in these cells with an efficacy that was comparable with that of its metabolic precursor δ-tocotrienol and higher than those of α-tocopherol metabolites. Unlike for genistein and the other vitamin E compounds, the GA-induced restoration of ApoE efflux was not affected by pharmacological inhibition of PPARγ activity, and specific activation of pregnane X receptor (PXR) was observed together with ApoE and multidrug resistance protein 1 (MDR1) membrane transporter up-regulation in both the mouse astrocytes and brain tissue. These effects of GA were associated with reduced Aβ deposition in the brain of TgCRND8 mice, a transgenic AD model. In conclusion, GA holds potential for preventing Aβ oligomerization and deposition in the brain. The mechanistic aspects of GA's properties appear to be distinct from those of other vitamin E metabolites and of genistein., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest., (© 2020 Marinelli et al.)

Bone remodeling and repair require osteogenic cells to reach the sites that need to be rebuilt, indicating that stimulation of osteoblast migration could be a promising osteoanabolic strategy. We showed that purified δ-tocotrienol (δ-TT, 10 μg/mL), isolated from commercial palm oil ( Elaeis guineensis ) fraction, stimulates the migration of both MC3T3-E1 osteoblast-like cells and primary human bone marrow mesenchymal stem cells (BMSC) as detected by wound healing assay or Boyden chamber assay respectively. The ability of δ-TT to promote MC3T3-E1 cells migration is dependent on Akt phosphorylation detected by Western blotting and involves Wnt/β-catenin signalling pathway activation. In fact, δ-TT increased β-catenin transcriptional activity, measured using a Nano luciferase assay and pretreatment with procaine (2 µM), an inhibitor of the Wnt/β-catenin signalling pathway, reducing the wound healing activity of δ-TT on MC3T3-E1 cells. Moreover, δ-TT treatment increased the expression of β-catenin specific target genes, such as Osteocalcin and Bone Morphogenetic Protein-2, involved in osteoblast differentiation and migration, and increased alkaline phosphatase and collagen content, osteoblast differentiation markers. The ability of δ-TT to enhance the recruitment of BMSC, and to promote MC3T3-E1 differentiation and migratory behavior, indicates that δ-TT could be considered a promising natural anabolic compound.

Studies on tocotrienols have progressively revealed the benefits of these vitamin E isoforms on human health. Beta-tocotrienol (beta-T3) is known to be less available in nature compared to other vitamin E members, which may explain the restricted number of studies on beta-T3. In the present study, we aim to investigate the anti-proliferative effects and the pro-apoptotic mechanisms of beta-T3 on two human breast adenocarcinoma cell lines MDA-MB-231 and MCF7. To assess cell viability, both cell lines were incubated for 24 and 48 h, with different concentrations of beta-T3 and gamma-T3, the latter being a widely studied vitamin E isoform with potent anti-cancerous properties. Cell cycle progression and apoptosis induction upon treatment with various concentrations of the beta-T3 isoform were assessed. The effect of beta-T3 on the expression level of several apoptosis-related proteins p53, cytochrome C, cleaved-PARP-1, Bax, Bcl-2, and caspase-3, in addition to key cell survival proteins p-PI3K and p-GSK-3 α/β was determined using western blot analysis. Beta-tocotrienol exhibited a significantly more potent anti-proliferative effect than gamma-tocotrienol on both cell lines regardless of their hormonal receptor status. Beta-T3 induced a mild G1 arrest on both cell lines, and triggered a mitochondrial stress-mediated apoptotic response in MDA-MB-231 cells. Mechanistically, beta-T3's anti-neoplastic activity involved the downregulation of phosphorylated PI3K and GSK-3 cell survival proteins. These findings suggest that vitamin E beta-T3 should be considered as a promising anti-cancer agent, more effective than gamma-T3 for treating human breast cancer and deserves to be further studied to investigate its effects in vitro and on other cancer types.

D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS1000) is the most active water-soluble derivative of vitamin E and has been widely used as a carrier of solvents, plasticizers, emulsifiers, absorbent agents and refractory drug delivery systems. However, its anti-hepatocellular carcinoma (HCC) properties have not been explored. HCC cells were treated with different concentrations of TPGS1000. Cell survival was tested by CCK8 assay, and cell migration was tested by wound healing and Transwell assay. EdU staining verified cell proliferation, and signalling pathway was assayed by Western blot analysis. The BALB/c-nu mouse xenograft model was established to test HCC cell growth in vivo . In vitro TPGS1000 significantly inhibited the viability and mobility of HCC cells (HepG2, Hep3B and Huh7) in a dose-dependent manner. Cell cycle analysis indicated that TPGS1000 treatment arrested the HCC cell cycle in the G0/G1 phase, and induction of cell apoptosis was confirmed by TUNEL and Annexin V-7-AAD staining. Further pharmacological analysis indicated that collapse of the transmembrane potential of mitochondria, increased ROS generation, PARP-induced cell apoptosis and FoxM1-p21-mediated cell cycle arresting, were involved in the anti-HCC activity of TPGS1000. Moreover, treatment in vivo with TPGS1000 effectively impaired the growth of HCC xenografts in nude mice.

Prostate cancer is the second most commonly diagnosed cancer in men, and metastatic prostate cancer is currently incurable. Prostate cancer frequently becomes resistant to standard of care treatments, and the administration of chemotherapeutic drugs is often accompanied by toxic side effects. Combination therapy is one tool that can be used to combat therapeutic resistance and drug toxicity. Vitamin E (VE) compounds and analogs have been proposed as potential non-toxic chemotherapeutics. Here we modeled combination therapy using mixture design response surface methodology (MDRSM), a statistical technique designed to optimize mixture compositions, to determine whether combinations of three chemotherapeutic agents: γ-tocotrienol (γ-T3), α-tocopherol ether acetate (α-TEA), and docetaxel (DOC), would prove more effective than docetaxel alone in the treatment of human prostate cancer cells. Response surfaces were generated for cell viability, and the optimal treatment combination for reducing cell viability was calculated. We found that a combination of 20 µM γ-T3, 30 µM α-TEA, and 25 nm DOC was most effective in the treatment of PC-3 cells. We also found that the combination of γ-T3 and α-TEA with DOC decreased the amount of DOC required to reduce cell viability in PC-3 cells and ameliorated therapeutic resistance in DOC-resistant PC-3 cells.

Countercurrent chromatography (CCC) was used for the enrichment of α-tocodienol (α-T 2 ), a rare vitamin E-related minor compound previously tentatively detected in palm oil. Hitherto, only one isomer has been mentioned to occur at traces in palm oil. However, CCC fractionation followed by GC/MS measurements of all fractions resulted in the detection of two α-T 2 isomers in five different palm oil vitamin E dietary supplement capsules. Five repetitive CCC separations of ~ 1 g sample and additional purification steps by column chromatography provided ~ 2 mg of two equally abundant α-T 2 isomers with a purity of ~ 85%. The positions of the double bonds in the alkyl side chain could be assigned by means of two characteristic chemical shifts in the 1 H NMR spectrum. Accordingly, the structures of the α-T 2 isomers were 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridec-3,11-dienyl)chroman-6-ol (double bonds in 3',11'-position) and 2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridec-7,11-dienyl)chroman-6-ol (double bonds in 7',11'-position). Natural occurrence of both isomers was proven by GC/MS screening of crude palm oil after saponification and CCC separation. Moreover, GC/MS analysis allowed the tentative assignment of γ-tocomonoenol (γ-T 1 ) and β-tocomonoenol (β-T 1 ) as trace compounds in palm oil.

Acute myeloid leukemia (AML) is a blood cancer characterized by the formation of faulty defective myelogenous cells with morphological heterogeneity and cytogenic aberrations leading to a loss of their function. In an attempt to find an effective and safe AML treatment, vitamin E derivatives, including tocopherols were considered as potential anti-tumor compounds. Recently, other isoforms of vitamin E, namely tocotrienols have been proposed as potential potent anti-cancerous agents, displaying promising therapeutic effects in different cancer types. In this study we evaluated the anti-cancerous effects of γ-tocotrienol, on AML cell lines in vitro . For this purpose, AML cell lines incubated with γ-tocotrienol were examined for their viability, cell cycle status, apoptotic cell death, DNA fragmentation, production of reactive oxygen species and expression of proapoptotic proteins. Our results showed that γ-tocotrienol exhibits time and dose-dependent anti-proliferative, pro-apoptotic and antioxidant effects on U937 and KG-1 cell lines, through the upregulation of proteins involved in the intrinsic apoptotic pathway., Competing Interests: The authors declare no conflicts of interest.

The plant Garcinia kola is used in African ethno-medicine to treat various oxidation- and inflammation-related diseases but its bioactive compounds are not well characterized. Garcinoic acid (GA) is one of the few phytochemicals that have been isolated from Garcinia kola. We investigated the anti-inflammatory potential of the methanol extract of Garcinia kola seeds (NE) and purified GA, as a major phytochemical in these seeds, in lipopolysaccharide (LPS)-activated mouse RAW264.7 macrophages and its anti-atherosclerotic potential in high fat diet fed ApoE -/- mice. This study outlines an optimized procedure for the extraction and purification of GA from Garcinia kola seeds with an increased yield and a purity of >99%. We found that LPS-induced upregulation of iNos and Cox2 expression, and the formation of the respective signaling molecules nitric oxide and prostanoids, were significantly diminished by both the NE and GA. In addition, GA treatment in mice decreased intra-plaque inflammation by attenuating nitrotyrosinylation. Further, modulation of lymphocyte sub-populations in blood and spleen have been detected, showing immune regulative properties of GA. Our study provides molecular insights into the anti-inflammatory activities of Garcinia kola and reveals GA as promising natural lead for the development of multi-target drugs to treat inflammation-driven diseases., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

This study evaluated the preclinical activity of δ-tocotrienol (DT3), a bioactive form of vitamin E, in the inhibition of colorectal cancer growth and development in vitro and in vivo DT3 is the most bioactive isomer of vitamin E in inhibiting growth of colorectal cancer cells. However, it had little effect on the proliferation of normal colon mucosal cells NCM460. In HCT-116 and SW-620 colorectal cancer cells, DT3 (50 μmol/L) significantly inhibited malignant transformation ( P < 0.02, P < 0.001), cell migration ( P < 0.02, P < 0.05), and invasion ( P < 0.05, P < 0.01) compared with vehicle. DT3 inhibited markers for epithelial (E-cadherin) to mesenchymal (vimentin) transition, metastasis (matrix metalloproteinase 9), angiogenesis VEGF, inflammation (NF-κB), and Wnt signaling (β-catenin) compared with vehicle in colorectal cancer cells. DT3 induced apoptosis selectively in colorectal cancer cells (SW-620 cells, HCT-116 cells, and HT-29) without affecting the normal colon cells. In the azoxymethane-induced colorectal carcinogenesis model in rats, DT3 (200 mg/kg orally twice a day) for 20 weeks significantly inhibited colorectal polyps by 70% and colorectal cancer by almost 99% compared with the vehicle treatment group ( P < 0.02, P < 0.001), and the cancer inhibition effect was more potent than sulindac (50%). Taken together, these data demonstrate that DT3 is a potential chemopreventive agent in colorectal cancer, warranting further investigation into its clinical use in the prevention and treatment of colorectal cancer., (©2019 American Association for Cancer Research.)

Omega 3-docosahexaenoic acid (DHA) and vitamin E Delta-tocotrienol (Delta-T3) are extensively studied as protective nutrients against cancer development. Little is known about the biological mechanisms targeted by these bioactive molecules on lipid droplet (LD) biogenesis, an important breast cancer aggressiveness marker, and the occurrence of lipophagy in breast cancer cells. The aim of this study was to investigate the effect of DHA, Delta-T3 and DHA plus Delta-T3 co-treatment in LD biogenesis and lipophagy process in triple negative breast cancer cell line MDA-MB-231. Cells were treated with 50 μM DHA and/or 5 μM Delta-T3. Our results demonstrated that DHA can trigger an increase in LD biogenesis and co-treatment with Delta-T3 was able to reduce this LD biogenesis. In addition, we showed that a higher cytoplasmic LD content is associated with a higher breast cancer cells malignance and proliferation. Reduction of cytoplasmic LD content by silencing ADRP (adipose differentiation-related protein), a structural LD protein, also decreased cell proliferation in MDA-MB-231 cells. Treatment with DHA and Delta-T3 alone or co-treatment did not reduce cell viability. Moreover, we showed here that DHA can trigger lipophagy in MDA-MB-231 cells and DHA plus Delta-T3 co-treatment was able to enhance this lipophagy process. Our findings demonstrated that co-treatment with DHA plus Delta-T3 in MDA-MB-231 cells could reduce LD biogenesis and potentiate lipophagy in these cells, possibly having a positive impact to inhibit breast cancer malignancy. Therefore, suitable doses of DHA and Delta-T3 vitamin E isoform supplementation can be a prominent tool in therapeutic treatments against breast cancer., Competing Interests: The authors declare no conflict of interest.

Objectives: Prostate cancer, after the phase of androgen dependence, may progress to the castration-resistant prostate cancer (CRPC) stage, with resistance to standard therapies. Vitamin E-derived tocotrienols (TTs) possess a significant antitumour activity. Here, we evaluated the anti-cancer properties of δ-TT in CRPC cells (PC3 and DU145) and the related mechanisms of action., Materials and Methods: MTT, Trypan blue and colony formation assays were used to assess cell viability/cell death/cytotoxicity. Western blot, immunofluorescence and MTT analyses were utilized to investigate apoptosis, ER stress and autophagy. Morphological changes were investigated by light and transmission electron microscopy., Results: We demonstrated that δ-TT exerts a cytotoxic/proapoptotic activity in CRPC cells. We found that in PC3 cells: (a) δ-TT triggers both the endoplasmic reticulum (ER) stress and autophagy pathways; (b) autophagy induction is related to the ER stress, and this ER stress/autophagy axis is involved in the antitumour activity of δ-TT; in autophagy-defective DU145 cells, only the ER stress pathway is involved in the proapoptotic effects of δ-TT; (c) in both CRPC cell lines, δ-TT also induces an intense vacuolation prevented by the ER stress inhibitor salubrinal and the protein synthesis inhibitor cycloheximide, together with increased levels of phosphorylated JNK and p38, supporting the induction of paraptosis by δ-TT., Conclusions: These data demonstrate that apoptosis, involving ER stress and autophagy (in autophagy positive PC3 cells), and paraptosis are involved in the anti-cancer activity of δ-TT in CRPC cells., (© 2019 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Emerging evidence suggests that gamma-tocotrienol (γ-T3), a vitamin E isomer, has potent anti-cancer properties against a wide-range of cancers. γ-T3 not only inhibited the growth and survival of cancer cells in vitro, but also suppressed angiogenesis and tumour metastasis under in vivo conditions. Recently, γ-T3 was found to target cancer stem cells (CSCs), leading to suppression of tumour formation and chemosensitisation. Despite its promising anti-cancer potential, the exact mechanisms responsible for the effects of γ-T3 are still largely unknown. Here, we report the identification of Ang-1 (Angiopoietin-1)/Tie-2 as a novel γ-T3 downstream target. In prostate cancer cells, γ-T3 treatment leads to the suppression of Ang-1 at both the mRNA transcript and protein levels. Supplementing the cells with Ang-1 was found to protect them against the anti-CSC effect of γ-T3. Intriguingly, inactivation of Tie-2, a member receptor that mediates the effect of Ang-1, was found to significantly enhance the cytotoxic effect of γ-T3 through activation of AMP-activated protein kinase (AMPK) and subsequent interruption of autophagy. Our results highlighted the therapeutic potential of using γ-T3 in combination with a Tie-2 inhibitor to treat advanced prostate cancer.

Protective action by annatto-derived delta-tocotrienol (δ-TCT) and soy-derived alpha-tocopherol (α-TOC) through the regulation of the PI3K/Akt-cyclin D1 pathway against nicotine-induced DNA damage is the focus of the present study. Nicotine, which has been widely reported to have numerous adverse effects on the reproductive system, was used as a reproductive toxicant. 48 female balb/c mice (6⁻8 weeks) (23⁻25 g) were randomly divided into eight groups (Grp.1⁻Grp.8; n = 6) and treated with either nicotine or/and annatto δ-TCT/soy α-TOC for seven consecutive days. On Day 8, the females were superovulated and mated before euthanization for embryo collection (46 h post-coitum). Fifty 2-cell embryos from each group were used in gene expression analysis using Affymetrix QuantiGene Plex2.0 assay. Findings indicated that nicotine (Grp.2) significantly decreased ( p < 0.05) the number of produced 2-cell embryos compared to the control (Grp.1). Intervention with mixed annatto δ-TCT (Grp.3) and pure annatto δ-TCT (Grp.4) significantly increased the number of produced 2-cell embryos by 127% and 79%, respectively compared to Grp.2, but these were lower than Grp.1. Concurrent treatment with soy α-TOC (Grp.5) decreased embryo production by 7%. Supplementations with δ-TCT and α-TOC alone (Grp.6-Grp.8) significantly increased ( p < 0.05) the number of produced 2-cell embryos by 50%, 36%, and 41%, respectively, compared to control (Grp.1). These results were found to be associated with alterations in the PI3K/Akt-Cyclin D1 genes expressions, indicating the inhibitory effects of annatto δ-TCT and soy α-TOC against nicotinic embryonic damage. To our knowledge, this is the first attempt in studying the benefits of annatto δ-TCT on murine preimplantation 2-cell embryos.

Parkinson's disease (PD) is a common progressive neurodegenerative disease. It has been reported that oxidative stress contributes, at least in part, to its pathogenesis. Although dietary epidemiological studies suggest that sufficient intake of vitamin E may prevent the onset of PD, antioxidative therapy for PD with exogenous antioxidants involving α-tocopherol has not been successful in the clinical setting thus far. In recent years, the non-antioxidant activities of vitamin E have been given attention to. In the present study, to determine the antioxidant-independent cytoprotective activity of vitamin E, we investigated whether tocotrienols (T3s), another members of vitamin E family, exhibit the neuroprotective effect in cell and mouse models of PD independently of their antioxidant activities. Treatment with T3s, especially γ- and δ-T3s, exhibited cytoprotective effects via activation of PI3K/Akt signaling pathway in a cellular PD model. We also identified estrogen receptor (ER) β as an upstream mediator of PI3K/Akt signaling and demonstrated the direct binding of T3 to ERβ in vitro. Silencing expression of caveolin suppressed the cytoprotective effects of T3, indicating that caveola formation plays an important role in the cytoprotection by T3 via ERβ/PI3K/Akt signaling pathway. Thus it has been shown that T3 exerts cytoprotective function by a novel mechanism, which includes membrane ERβ/PI3K/Akt signaling via caveola formation as well as its antioxidant activity. Furthermore, we revealed that δ-T3 treatment relieved PD-related symptoms in PD model mice. These results suggest that T3 elicits the cytoprotective effects via ERβ/PI3K/Akt signaling pathway in cellular and murine PD models.

Background: Replicative senescence of human diploid fibroblasts (HDFs) has been used as a model to study mechanisms of cellular aging. Gamma-tocotrienol (γT3) is one of the members of vitamin E family which has been shown to increase proliferation of senescent HDFs. However, the modulation of protein expressions by γT3 in senescent HDFs remains to be elucidated. Therefore, this study aimed to determine the differentially expressed proteins (DEPs) in young and senescent HDFs; and in vehicle- and γT3-treated senescent HDFs using label-free quantitative proteomics., Methods: Whole proteins were extracted and digested in-gel with trypsin. Peptides were detected by Orbitrap liquid chromatography mass spectrometry. Mass spectra were identified and quantitated by MaxQuant software. The data were further filtered and analyzed statistically using Perseus software to identify DEPs. Functional annotations of DEPs were performed using Panther Classification System., Results: A total of 1217 proteins were identified in young and senescent cells, while 1218 proteins in vehicle- and γT3-treated senescent cells. 11 DEPs were found in young and senescent cells which included downregulation of platelet-derived growth factor (PDGF) receptor beta and upregulation of tubulin beta-2A chain protein expressions in senescent cells. 51 DEPs were identified in vehicle- and γT3-treated senescent cells which included upregulation of 70 kDa heat shock protein, triosephosphate isomerase and malate dehydrogenase protein expressions in γT3-treated senescent cells., Conclusions: PDGF signaling and cytoskeletal structure may be dysregulated in senescent HDFs. The pro-proliferative effect of γT3 on senescent HDFs may be mediated through the stimulation of cellular response to stress and carbohydrate metabolism. The expressions and roles of these proteins in relation to cellular senescence are worth further investigations. Data are available via ProteomeXchange with identifier PXD009933.

δ-Tocotrienol, an important component of vitamin E, has been reported to possess some physiological functions, such as anticancer and anti-inflammation, however their molecular mechanisms are not clear. In this study, δ-tocotrienol was isolated and purified from rice bran. The anti-inflammatory effect and mechanism of δ-tocotrienol against lipopolysaccharides (LPS) activated pro-inflammatory mediator expressions in RAW264.7 cells were investigated. Results showed that δ-tocotrienol significantly inhibited LPS-stimulated nitric oxide (NO) and proinflammatory cytokine (TNF-α, IFN-γ, IL-1β and IL-6) production and blocked the phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular regulated protein kinases 1/2 (ERK1/2). δ-Tocotrienol repressed the transcriptional activations and translocations of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1), which were closely related with downregulated cytokine expressions. Meanwhile, δ-tocotrienol also affected the PPAR signal pathway and exerted an anti-inflammatory effect. Taken together, our data showed that δ-tocotrienol inhibited inflammation via mitogen-activated protein kinase (MAPK) and peroxisome proliferator-activated receptor (PPAR) signalings in LPS-stimulated macrophages.

Background: δ-Tocotrienol is a naturally occurring proteasome inhibitor, which has the capacity to inhibit proliferation and induce apoptosis in several cancer cells obtained from several organs of humans, and other cancer cell lines. Moreover, results of plasma total mRNAs after δ-tocotrienol feeding to hepatitis C patients revealed significant inhibition in the expression of pro-inflammatory cytokines (TNF-α, VCAM1, proteasome subunits) and induction in the expression of ICAM1 and IFN-γ after post-treatment. This down-regulation of proteasome subunits leads to autophagy, apoptosis of immune cells and several genes. The present study describes RNA-sequence analysis of plasma total mRNAs obtained from δ-tocotrienol treatment of hepatitis C patients on gene expression regulated by proteasome., Methods: Pooled specimens of plasma total mRNAs of pre-dose versus post-dose of δ-tocotrienol treatment of hepatitis C patients were submitted to RNA-sequence analyses. The data based on > 1 and 8-fold expression changes of 2136 genes were uploaded into "Ingenuity Pathway Analyses (IPA)" for core analysis, which describes possible canonical pathways, upstream regulators, diseases and functional metabolic networks., Results: The IPA of "molecules" indicated fold change in gene expression of 953 molecules, which covered several categories of biological biomarkers. Out of these, gene expression of 220 related to present study, 12 were up-regulated, and 208 down-regulated after δ-tocotrienol treatment. The gene expression of transcription regulators (ceramide synthase 3 and Mohawk homeobox) were up-regulated, and gene expression of 208 molecules were down-regulated, involved in several biological functions (HSP90AB1, PSMC3, CYB5R4, NDUFB1, CYP2R1, TNFRF1B, VEGFA, GPR65, PIAS1, SFPQ, GPS2, EIF3F, GTPBP8, EIF4A1, HSPA14, TLR8, TUSSC2). IPA of "causal network" indicated gene regulators (676), in which 76 down-regulated (26 s proteasomes, interleukin cytokines, and PPAR-ligand-PPA-Retinoic acid-RXRα, PPARγ-ligand-PPARγ-Retinoic acid-RARα, IL-21, IL-23) with significant P-values. The IPA of "diseases and functions" regulators (85) were involved with cAMP, STAT2, 26S proteasome, CSF1, IFNγ, LDL, TGFA, and microRNA-155-5p, miR-223, miR-21-5p. The IPA of "upstream analysis" (934) showed 57 up-regulated (mainly 38 microRNAs) and 64 gene regulators were down-regulated (IL-2, IL-5, IL-6, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-24, IL-27, IL-32), interferon β-1a, interferon γ, TNF-α, STAT2, NOX1, prostaglandin J2, NF-κB, 1κB, TCF3, and also miRNA-15, miRNA-124, miRNA-218-5P with significant activation of Z-Score (P < 0.05)., Conclusions: This is first report describing RNA-sequence analysis of δ-tocotrienol treated plasma total mRNAs obtained from chronic hepatitis C patients, that acts via multiple-signaling pathways without any side-effects. These studies may lead to development of novel classes of drugs for treatment of chronic hepatitis C patients.

Background: Ceramide synthesis and metabolism is a promising target in cancer drug development. γ-tocotrienol (GT3), a member of the vitamin E family, orchestrates multiple effects that ensure the induction of apoptosis in both, wild-type and RAS-mutated pancreatic cancer cells. Here, we investigated whether these effects involve changes in ceramide synthesis and transport., Methods: The effects of GT3 on the synthesis of ceramide via the de novo pathway, and the hydrolysis of sphingomyelin were analyzed by the expression levels of the enzymes serine palmitoyl transferase, ceramide synthase-6, and dihydroceramide desaturase, and acid sphingomyelinase in wild-type RAS BxPC3, and RAS-mutated MIA PaCa-2 and Panc 1 pancreatic cancer cells. Quantitative changes in ceramides, dihydroceramides, and sphingomyelin at the cell membrane were detected by LCMS. Modulation of ceramide transport by GT3 was studied by immunochemistry of CERT and ARV-1, and the subsequent effects at the cell membrane was analyzed via immunofluorescence of ceramide, caveolin, and DR5., Results: GT3 favors the upregulation of ceramide by stimulating synthesis at the ER and the plasma membrane. Additionally, the conversion of newly synthesized ceramide to sphingomyelin and glucosylceramide at the Golgi is prevented by the inhibition of CERT. Modulation ARV1 and previously observed inhibition of the HMG-CoA pathway, contribute to changes in membrane structure and signaling functions, allows the clustering of DR5, effectively initiating apoptosis., Conclusions: Our results suggest that GT3 targets ceramide synthesis and transport, and that the upregulation of ceramide and modulation of transporters CERT and ARV1 are important contributors to the apoptotic properties demonstrated by GT3 in pancreatic cancer cells.

Background: Cancer is second most common cause of death in the United State. There are over 100 different types of cancer associated with different human organs, predominantly breast, liver, pancreas, prostate, colon, rectum, lung, and stomach. We have recently reported properties of pro-inflammatory (for treatment of various types of cancers), and anti-inflammatory (for cardiovascular disease and diabetes) compounds. The major problem associated with development of anticancer drugs is their lack of solubility in aqueous solutions and severe side effects in cancer patients. Therefore, the present study was carried out to check anticancer properties of selected compounds, mostly aqueous soluble, in cancer cell lines from different organs., Methods: The anticancer properties, anti-proliferative, and pro-apoptotic activity of novel naturally occurring or FDA approved, nontoxic, proteasome inhibitors/activators were compared. In addition to that, effect of δ-tocotrienol on expression of proteasome subunits (X, Y, Z, LMP7, LMP2, LMP10), ICAM-1, VCAM-1, and TNF-α using total RNAs derived from plasmas of hepatitis C patients was investigated., Results: Our data demonstrated that following compounds are very effective in inducing apoptosis of cancer cells: Thiostrepton, dexamethasone, 2-methoxyestradiol, δ-tocotrienol, quercetin, amiloride, and quinine sulfate have significant anti-proliferation properties in Hela cells (44% - 87%) with doses of 2.5-20 μM, compared to respective controls. Anti-proliferation properties of thiostrepton, 2-methoxyestradiol, δ-tocotrienol, and quercetin were 70% - 92%. However, thiostrepton, dexamethasone, 2-methoxyestradiol, δ-tocotrienol, quercetin, and quinine sulphate were effective in pancreatic, prostate, breast, lungs, melanoma, Β-lymphocytes, and T-cells (Jurkat: 40% to 95%) compared to respective controls. In lung cancer cells, these compounds were effective between 5 and 40 μM. The IC 50 values of anti-proliferation properties of thiostrepton in most of these cell lines were between doses of 2.5-5 μM, dexamethasone 2.5-20 μM, 2-methoxyestradiol 2.5-10 μM, δ-tocotrienol 2.5-20 μM, quercetin 10-40 μM, and (-) Corey lactone 40-80 μM. In hepatitis C patients, δ-tocotrienol treatment resulted in significant decrease in the expression of pro-inflammatory cytokines., Conclusions: These data demonstrate effectiveness of several natural-occurring compounds with anti-proliferative properties against cancer cells of several organs of humans. Thiostrepton, dexamethasone, 2-methoxyestradiol, δ-tocotrienol and quercetin are very effective for apoptosis of cancer cells in liver, pancreas, prostate, breast, lung, melanoma, Β-lymphocytes and T-cells. The results have provided an opportunity to test these compounds either individually or in combination as dietary supplements in humans for treatment of various types of cancers.

Background/aim: A hallmark of the progression of prostate cancer to advanced disease is the acquisition of androgen-independent growth. This malignant phenotype is characterized by resistance to conventional treatments and predisposes to formation of hypoxic regions containing stem-like cancer cells. Unfortunately, an effective therapy to target prostate cancer stem cells under hypoxia has not yet been established. In this report, we studied whether δ-tocotrienol (T3), a vitamin E family member that has exhibited the most potent anti-cancer activity, could suppress the survival of prostate cancer stem-like cells., Materials and Methods: PC3 stem-like cells were isolated from PC3 parental cells using a three-dimensional culture system. The stemness of the isolated PC3 stem-like cells was confirmed by evaluation of resistance to an anticancer agent (docetaxel) and tumor formation capacity in a xenograft model. The effects of δ-T3 on PC3 stem-like cells under a hypoxia condition were examined by WST-8 (cell viability), real-time reverse transcription-polymerase chain reaction (PCR) and western blotting., Results: δ-T3 demonstrated a cytotoxic effect on prostate cancer stem-like cells in a dose dependent manner and a reduction in the protein levels of hypoxia-inducible factor (HIF)-1α and HIF-2α. Additionally, a specific inhibitor toward HIF-1α induced cytotoxicity on PC3 cells, but selective inhibition of HIF-2α had no effect., Conclusion: Overall, these results suggest that δ-T3 could inhibit the survival of prostate cancer stem-like cells under hypoxia, primarily through the inactivation of HIF-1α signaling., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aims: Non-alcoholic fatty liver disease (NAFLD) is a growing public health problem worldwide and is associated with increased morbidity and mortality. Currently, there is no definitive treatment for this disease. δ-Tocotrienol has potent anti-inflammatory and antioxidant properties and may reduce liver injury in NAFLD. The present study aims to evaluate the efficacy and safety of δ-tocotrienol in the treatment of NAFLD., Materials and Methods: The present study was a randomized, double-blind, placebo-controlled pilot study conducted in patients aged > 20 years, belonging to both sexes, having ultrasound-proven fatty liver disease, having a fatty liver index (FLI) of ≥ 60, and persistent elevation of alanine transaminase. A total of 71 patients were assigned to receive either oral δ-tocotrienol (n=35, 300 mg twice daily) or placebo (n=36) for 12 weeks. At the baseline and at the end of the study, clinical and biochemical parameters, including lipid profile, liver function tests, high-sensitivity C-reactive protein (hs-CRP), and malondialdehyde (MDA) were measured. Body mass index and FLI were calculated, and ultrasound grading of hepatic steatosis was performed., Results: Out of 71 enrolled patients, 64 patients, 31 in the δ-tocotrienol group and 33 in the placebo group, completed the study. After 12 weeks of supplementation, δ-tocotrienol showed greater efficacy than placebo by decreasing serum aminotransferases, hs-CRP, MDA, and FLI score (p<0.001). However, it did not improve hepatic steatosis on ultrasound examination. No adverse effects were reported., Conclusion: δ-Tocotrienol was safe, and it effectively improved aminotransferase levels and inflammatory and oxidative stress markers in patients with NAFLD. Large-scale randomized clinical trials are warranted to further support these findings.

The prognosis of metastatic melanoma is very poor, due to the development of drug resistance. Cancer stem cells (CSCs) may play a crucial role in this mechanism, contributing to disease relapse. We first characterized CSCs in melanoma cell lines. We observed that A375 (but not BLM) cells are able to form melanospheres and show CSCs traits: expression of the pluripotency markers SOX2 and KLF4, higher invasiveness and tumor formation capability in vivo with respect to parental adherent cells. We also showed that a subpopulation of autofluorescent cells expressing the ABCG2 stem cell marker is present in the A375 spheroid culture. Based on these data, we investigated whether δ-TT might target melanoma CSCs. We demonstrated that melanoma cells escaping the antitumor activity of δ-TT are completely devoid of the ability to form melanospheres. In contrast, cells that escaped vemurafenib treatment show a higher ability to form melanospheres than control cells. δ-TT also induced disaggregation of A375 melanospheres and reduced the spheroidogenic ability of sphere-derived cells, reducing the expression of the ABCG2 marker. These data demonstrate that δ-TT exerts its antitumor activity by targeting the CSC subpopulation of A375 melanoma cells and might represent a novel chemopreventive/therapeutic strategy against melanoma.

Photosynthetic organisms such as plants, algae and some cyanobacteria synthesize tocochromanols, a group of compounds that encompasses tocopherols and tocotrienols and that exhibits vitamin E activity in animals. While most vitamin E biosynthetic genes have been identified in plant genomes, regulatory genes controlling tocopherol accumulation are currently unknown. We isolated by forward genetics Arabidopsis enhanced vitamin E (eve) mutants that overaccumulate the classic tocopherols and plastochromanol-8, and a tocochromanol unknown in this species. We mapped eve1 and eve4, and identified the unknown Arabidopsis tocochromanol by using a combination of analytical tools. In addition, we determined its biosynthetic pathway with a series of tocochromanol biosynthetic mutants and transgenic lines. eve1 and eve4 are two seed lipid mutants affecting the WRINKLED1 (WRI1) and ACYL-COA:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) genes, respectively. The unknown tocochromanol is 11'-12' γ-tocomonoenol, whose biosynthesis is VITAMIN E 1 (VTE1) - and VTE2-dependent and is initiated by the condensation of homogentisate (HGA) and tetrahydrogeranylgeranyl pyrophosphate. This study identifies the first two regulatory genes, WRI1 and DGAT1, that control the synthesis of all tocochromanol forms in seeds, and shows the existence of a metabolic trade-off between lipid and tocochromanol metabolisms. Moreover, it shows that Arabidopsis possesses a tocomonoenol biosynthetic pathway that competes with tocopherol synthesis., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

The development of radiation countermeasures for acute radiation syndrome (ARS) has been underway for the past six decades, leading to the identification of multiple classes of radiation countermeasures. However, to date, only two growth factors (Neupogen and Neulasta) have been approved by the United States Food and Drug Administration (US FDA) for the mitigation of hematopoietic acute radiation syndrome (H-ARS). No radioprotector for ARS has been approved by the FDA yet. Gamma-tocotrienol (GT3) has been demonstrated to have radioprotective efficacy in murine as well as nonhuman primate (NHP) models. Currently, GT3 is under advanced development as a radioprotector that can be administered prior to radiation exposure. We are studying this agent for its safety profile and efficacy using the NHP model. In this study, we analyzed global metabolomic and lipidomic changes using ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) in serum samples of NHPs administered GT3. Our study, using 12 NHPs, demonstrates that alterations in metabolites manifest only 24 h after GT3 administration. Furthermore, metabolic changes are associated with transient increase in the bioavailability of antioxidants, including lactic acid and cholic acid and anti-inflammatory metabolites 3 deoxyvitamin D3, and docosahexaenoic acid. Taken together, our results show that the administration of GT3 to NHPs causes metabolic shifts that would provide an overall advantage to combat radiation injury. This initial assessment also highlights the utility of metabolomics and lipidomics to determine the underlying physiological mechanisms involved in the radioprotective efficacy of GT3., Competing Interests: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

The synthesis of complex organic compounds usually relies on controlling the reactions of the functional groups. In recent years, it has become possible to carry out reactions directly on the C-H bonds, previously considered to be unreactive. One of the major challenges is to control the site-selectivity because most organic compounds have many similar C-H bonds. The most well developed procedures so far rely on the use of substrate control, in which the substrate has one inherently more reactive C-H bond or contains a directing group or the reaction is conducted intramolecularly so that a specific C-H bond is favoured. A more versatile but more challenging approach is to use catalysts to control which site in the substrate is functionalized. p450 enzymes exhibit C-H oxidation site-selectivity, in which the enzyme scaffold causes a specific C-H bond to be functionalized by placing it close to the iron-oxo haem complex. Several studies have aimed to emulate this enzymatic site-selectivity with designed transition-metal catalysts but it is difficult to achieve exceptionally high levels of site-selectivity. Recently, we reported a dirhodium catalyst for the site-selective functionalization of the most accessible non-activated (that is, not next to a functional group) secondary C-H bonds by means of rhodium-carbene-induced C-H insertion. Here we describe another dirhodium catalyst that has a very different reactivity profile. Instead of the secondary C-H bond, the new catalyst is capable of precise site-selectivity at the most accessible tertiary C-H bonds. Using this catalyst, we modify several natural products, including steroids and a vitamin E derivative, indicating the applicability of this method of synthesis to the late-stage functionalization of complex molecules. These studies show it is possible to achieve site-selectivity at different positions within a substrate simply by selecting the appropriate catalyst. We hope that this work will inspire the design of even more sophisticated catalysts, such that catalyst-controlled C-H functionalization becomes a broadly applied strategy for the synthesis of complex molecules.

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., (© 2017 American Society for Nutrition.)

There are six tocol analogs present in palm oil, namely α-tocopherol (α-T), α-tocomonoenol (α-T₁), α-tocotrienol (α-T₃), γ-tocotrienol (γ-T₃), β-tocotrioenol (β-T₃) and δ-tocotrienol (δ-T₃). These analogs were difficult to separate chromatographically due to their similar structures, physical and chemical properties. This paper reports on the effect of pressure and injection solvent on the separation of the tocol analogs in palm oil. Supercritical CO₂ modified with ethanol was used as the mobile phase. Both total elution time and resolution of the tocol analogs decreased with increased pressure. Ethanol as an injection solvent resulted in peak broadening of the analogs within the entire pressure range studied. Solvents with an eluent strength of 3.4 or less were more suitable for use as injecting solvents., Competing Interests: The authors declare no conflict of interest.

Background: γ-Tocotrienol, a vitamin E isomer possesses pronounced in vitro anticancer activities. However, the in vivo potency has been limited by hardly achievable therapeutic levels owing to inefficient high-dose oral delivery which leads to subsequent metabolic degradation. Jerantinine A, an Aspidosperma alkaloid, originally isolated from Tabernaemontana corymbosa, has proved to possess interesting anticancer activities. However, jerantinine A also induces toxicity to non-cancerous cells., Purpose: We adopted a combinatorial approach with the joint application of γ-tocotrienol and jerantinine A at lower concentrations in order to minimize toxicity towards non-cancerous cells while improving the potency on brain cancer cells., Methods: The antiproliferative potency of individual γ-tocotrienol and jerantinine A as well as combined in low-concentration was firstly evaluated on U87MG cancer and MRC5 normal cells. Morphological changes, DNA damage patterns, cell cycle arrests and the effects of individual and combined low-concentration compounds on microtubules were then investigated. Finally, the potential roles of caspase enzymes and apoptosis-related proteins in mediating the apoptotic mechanisms were investigated using apoptosis antibody array, ELISA and Western blotting analysis., Results: Combinatorial study between γ-tocotrienol at a concentration range (0-24µg/ml) and fixed IC 20 concentration of jerantinine A (0.16µg/ml) induced a potent antiproliferative effect on U87MG cells and led to a reduction on the new half maximal inhibitory concentration of γ-tocotrienol (i.e. t IC 50 =1.29µg/ml) as compared to that of individual γ-tocotrienol (i.e. IC 50 =3.17µg/ml). A reduction on undesirable toxicity to MRC5 normal cells was also observed. G0/G1 cell cycle arrest was evident on U87MG cells receiving IC 50 of individual γ-tocotrienol and combined low-concentration compounds (1.29µg/ml γ-tocotrienol + 0.16µg/ml jerantinine A), whereas, a profound G2/M arrest was evident on cells treated with IC 50 of individual jerantinine A. Additionally, individual jerantinine A and combined compounds (except individual γ-tocotrienol) caused a disruption of microtubule networks triggering Fas- and p53-induced apoptosis mediated via the death receptor and mitochondrial pathways., Conclusions: These findings demonstrated that the combined use of lower concentrations of γ-tocotrienol and jerantinine A induced potent cytotoxic effects on U87MG cancer cells resulting in a reduction on the required individual concentrations and thereby minimizing toxicity of jerantinine A towards non-cancerous MRC5 cells as well as probably overcoming the high-dose limiting application of γ-tocotrienol. The multi-targeted mechanisms of action of the combination approach have shown a therapeutic potential against brain cancer in vitro and therefore, further in vivo investigations using a suitable animal model should be the way forward., (Copyright © 2017 Elsevier GmbH. All rights reserved.)