A RP-HPLC method for the simultaneous analysis of tocotrienol isoforms (TRF) and simvastatin (SIM) in SIM-TRF nanoparticles (NPs) was developed. Analytes were monitored by UV detection at 238 and 295 nm for SIM and TRF, respectively, using a gradient methanol/water elution. Calibration curves for TRF and SIM were linear over concentration range of 20-80 microg/mL and 1-10 microg/mL with correlation coefficients 0.9990 and 0.9991, respectively. The recovery of TRF and SIM from the NPs was in the range from 97.35 to 102.19% and from 92.71 to 104.35%, respectively. This developed method was successfully employed in quantifying both drugs in NPs for future use in cancer therapy.

The metabolism of gamma-tocotrienol (gamma-TE) and gamma-tocopherol (gamma-T) was investigated in human A549 cells and in rats. Similar to gamma-T, A549 cells metabolized gamma-TE to sulfated 9′-, 11′-, and 13′-carboxychromanol and their unconjugated counterparts. After 72-h incubation with the cells, 90% of long-chain carboxychromanols in the culture media from gamma-TE, but <45% from gamma-T, were in the sulfated form. The formation of these metabolites was further investigated in rats gavaged by gamma-TE at 10 or 50 mg/kg, gamma-T at 10 mg/kg, or tocopherol-stripped corn oil in controls. Six hours after a single dosing, the supplemented rats had increased plasma concentrations of 13′-carboxychromanol and sulfated 9′-, 11′-, 13′-carboxychromanol, whereas none of these metabolites were detectable in the controls. Sulfated 11′-carboxychromanol was the most abundant long-chain metabolite in gamma-TE-supplemented rats. Sulfatase/glucuronidase hydrolysis revealed for the first time that >88% 2-(beta-carboxyethyl)-6-hydroxychroman (gamma-CEHC), the terminal beta-oxidation metabolite, was in the conjugated form in the plasma. In all groups, conjugated gamma-CEHC accounted for >75% of total metabolites, whereas free CEHC was a minor metabolite. At 10 mg/kg, the plasma concentrations of total metabolites from gamma-TE-supplemented rats were higher (P < 0.05) than those from gamma-T-fed rats. These results demonstrate that in rats, conjugation such as sulfation occurs parallel to beta-oxidation in the liver and is quantitatively important to vitamin E metabolism. Conjugated long-chain carboxychromanols may be novel excreted metabolites during supplementation. Our data also provide in vivo evidence that gamma-TE is more extensively metabolized than gamma-T.

d-δ-Tocotrienol-mediated suppression of the proliferation of human PANC-1, MIA PaCa-2, and BxPC-3 pancreatic carcinoma cells

Hussein D, Mo H.

Pancreas. 2009 May;38(4):e124-36.

Objective: The rate-limiting activity of the mevalonate pathway, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, provides intermediates essential for growth. Competitive inhibitors of HMG CoA reductase, such as the statins, and down-regulators of reductase, such as the tocotrienols, suppress tumor growth. We evaluated the impact of d-delta-tocotrienol, the most potent vitamin E isomer, on human MIA PaCa-2 and PANC-1 pancreatic carcinoma cells and BxPC-3 pancreatic ductal adenocarcinoma cells.

Methods: Cell proliferation was measured by using CellTiter 96 Aqueous One Solution (Promega, Madison, Wis). Cell cycle distribution was determined by flow cytometry. Apoptosis was evaluated by Annexin V staining and fluorescence microscopy after dual staining with acridine orange and ethidium bromide.

Results: d-delta-Tocotrienol induced concentration-dependent suppression of cell proliferation with 50% inhibitory concentrations of 28 (6) micromol/L (MIA PaCa-2), 35 (7) micromol/L (PANC-1), and 35 (8) microL (BxPC-3), respectively. These effects are attributable to cell cycle arrest at the G1 phase and apoptosis. Mevalonate attenuated d-delta-tocotrienol-mediated growth inhibition. A physiologically attainable blend of d-delta-tocotrienol and lovastatin synergistically suppressed the proliferation of MIA PaCa-2 cells.

Conclusions: Suppression of mevalonate pathway activities, be it by modulators of HMG CoA reductase (statins, tocotrienols, and farnesol), farnesyl transferase (farnesyl transferase inhibitors), and/or mevalonate pyrophosphate decarboxylase (phenylacetate) activity, may have a potential in pancreatic cancer chemotherapy.

Tocopherols and tocotrienols have been simultaneously determined in food samples using a rapid and simple analytical method including pressurized liquid extraction (PLE) and LC with electrochemical detection. Separation was carried out on a Phenomenex Synergi 4 microm Hydro-RP 80A column, using a solution of 2.5 mM acetic acid/sodium acetate in methanol/water (99:1, v/v) as mobile phase at a flow rate of 1.0 mL/min. Column temperature was maintained at 30 degrees C. Detection was performed by coulometric detection at 500 mV except for (beta+gamma)-tocotrienol, in wheat and rye samples, which was at +350 mV. A palm oil containing a relatively large amount of gamma-tocotrienol and lower concentrations of alpha- and delta-tocotrienols and alpha- and gamma-tocopherols was used to provide reference retention times for the tocotrienols. Analyte quantification was performed using the external standard method. The calibration equations of tocopherols were used to quantify both tocopherols and their corresponding tocotrienols. The extraction recoveries obtained using the optimized PLE conditions were in the 80-114% range, with RSDs lower than 15%. The method was successfully applied to the determination of tocotrienols and tocopherols in cereal (wheat, rye, barley, maize and oat) and palm oil samples.

Gamma-tocotrienol ameliorates intestinal radiation injury and reduces vascular oxidative stress after total-body irradiation by an HMG-CoA reductase-dependent mechanism

Berbée M, Fu Q, Boerma M, Wang J, Kumar KS, Hauer-Jensen M.

Radiat Res. 2009 May;171(5):596-605.

Analogs of vitamin E (tocols) are under development as radioprophylactic agents because of their high efficacy and lack of toxicity. Gamma-tocotrienol (GT3) is of particular interest because, in addition to being an antioxidant, it also inhibits 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and accumulates to greater extent in endothelial cells than other tocols. We addressed in vivo whether HMG-CoA reductase inhibition contributes to the radioprotection conferred by GT3. Groups of mice were treated with vehicle, mevalonate (the product of the reaction catalyzed by HMG-CoA reductase), GT3 alone or GT3 in combination with mevalonate. Lethality and standard parameters of injury to the hematopoietic, intestinal and vascular/endothelial systems were assessed after exposure to total-body irradiation. GT3 improved postirradiation survival and decreased radiation-induced vascular oxidative stress, an effect that was reversible by mevalonate. GT3 also enhanced hematopoietic recovery, reduced intestinal radiation injury, and accelerated the recovery of soluble markers of endothelial function. These parameters were not reversed by mevalonate co-administration. Our data confirm GT3’s radioprophylactic properties against hematopoietic injury and, for the first time, demonstrate benefits in terms of protection against gastrointestinal and vascular injury. The radioprotective efficacy of GT3 against vascular injury is related to its properties as an HMG-CoA reductase inhibitor.

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A redox-silent analogue of tocotrienol acts as a potential cytotoxic agent against human mesothelioma cells

Kashiwagi K, Virgona N, Harada K, Kido W, Yano Y, Ando A, Hagiwara K, Yano T.

Life Sci. 2009 May 8;84(19-20):650-6.

Aims: Malignant mesothelioma is an aggressive cancer with no effective treatment options. A redox-silent analogue of alpha-tocotrienol, 6-O-carboxypropyl-alpha-tocotrienol (T3E) is a new potential anti-carcinogenic agent with less toxic effect on non-tumorigenic cells. Here, we evaluated the effect of T3E on killing of chemoresistant mesothelioma cell (H28). MAIN

Methods: The cytotoxic effect of T3E was evaluated by a WST-1 assay, and cell cycle and apoptosis analysis were done by FACS. Each signal molecule’s activity was determined by protein array and immunoblot analysis.

Key Findings: T3E effectively inhibited H28 cell growth at practical pharmacological concentrations (10-20 muM) without any effect on non-tumorigenic mesothelial cell (Met-5A). Inhibition of H28 cell growth by T3E mediated through G2/M arrest in cell cycle and induction of apoptosis. Protein array and immunoblot analyses revealed that T3E inhibited the activation of epidermal growth factor receptor (EGFR) via the inactivation of the Src family of protein tyrosine kinases (Src). However, the blockade of the EGFR signaling was not associated with the T3E-dependent H28 cell growth control. In addition to Src inactivation, T3E inhibited signal transduction and activation of transcription Stat3. A combination of an Src inhibitor, PP2, and a Stat3 inhibitor, AG490, induced G2/M arrest and enhanced apoptosis compared with PP2 alone. These results suggest that T3E suppresses H28 cell growth via the inhibition of Src activation and Src-independent Stat3 activation.

Significance: T3E can be a new effective therapeutic agent against chemoresistant mesothelioma cells.

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