Palm tocotrienols inhibit proliferation of murine mammary cancer cells and induce expression of interleukin-24 mRNA

Selvaduray KR, Radhakrishnan AK, Kutty MK, Nesaretnam K.

J Interferon Cytokine Res. 2010 Dec;30(12):909-16.

Several mechanisms have been postulated for the anticancer effects of tocotrienols. In this study, for the first time, the anticancer effect oftocotrienols is linked to increased expression of interleukin-24 (IL-24) mRNA, a cytokine reported to have antitumor effects in many cancer models.Tocotrienol isomers (α-T3, γ-T3, and δ-T3) and tocotrienol-rich fraction (TRF) inhibited the growth of the 4T1 murine mammary cancer cells (P < 0.05), with IC₅₀ values 8.99, 4.79, 3.73, and 8.63 μg/mL, respectively. Tumor incidence and tumor load in TRF-supplemented BALB/c mice was decreased by 57.1% and 93.6% (P < 0.05), respectively. The induction of the IL-24 mRNA in the 4T1 cells by vitamin E decreased in the following order: δ-T3 > γ-T3 > TRF > α-T3 > α-T, which was similar to their antiproliferative effects. The IL-24 mRNA levels in tumor tissues of BALB/c mice supplemented with TRF increased 2-fold when compared with control mice. Increased levels of IL-24 have been associated with inhibition of tumor growth and angiogenesis. Treatment of 4T1 cells with TRF and δ-T3 significantly decreased IL-8 and vascular endothelial growth factor mRNA levels. Hence, we report that tocotrienols have potent antiangiogenic and antitumor effects that is associated with increased levels of IL-24 mRNA.

Tocotrienols inhibit lipopolysaccharide-induced pro-inflammatory cytokines in macrophages of female mice

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

Lipids Health Dis. 2010 Dec 16;9:143.

Background: Inflammation has been implicated in cardiovascular disease, and the important role of proteasomes in the development of inflammation and other macrophage functions has been demonstrated. Tocotrienols are potent hypocholesterolemic agents that inhibit β-hydroxy-β-methylglutaryl coenzyme A reductase activity, which is degraded via the ubiquitin-proteasome pathway. Our objective was to evaluate the effect oftocotrienols in reducing inflammation. Lipopolysaccharide (LPS) was used as a prototype for inflammation in murine RAW 264.7 cells and BALB/c female mice.

Results: The present results clearly demonstrate that α-, γ-, or δ-tocotrienol treatments inhibit the chymotrypsin-like activity of 20 S rabbit muscle proteasomes (> 50%; P < 0.05). Chymotrypsin, trypsin, and post-glutamase activities were decreased > 40% (P < 0.05) with low concentrations (< 80 μM), and then increased gradually with concentrations of (80–640 μM) in RAW 264.7 whole cells. Tocotrienols showed 9–33% (P < 0.05) inhibitions in TNF-α secretion in LPS-stimulated RAW 264.7 cells. Results of experiments carried out in BALB/c mice demonstrated that serum levels of TNF-α after LPS treatment were also reduced (20–48%; P < 0.05) by tocotrienols with doses of 1 and 10 μg/kg, and a corresponding rise in serum levels of corticosterone (19–41%; P < 0.05) and adrenocorticotropic hormone (81–145%; P < 0.02) was observed at higher concentrations (40 μM). Maximal inhibition of LPS-induced TNF-α was obtained with δ-tocotrienol (10 μg/kg). Low concentrations of δ-Tocotrienols (< 20 μM) blocked LPS-induced gene expression of TNF-α, IL-1β, IL-6 and iNOS (> 40%), while higher concentrations (40 μM) increased gene expression of the latter in peritoneal macrophages (prepared from BALB/c mice) as compared to control group.

Conclusion: These results represent a novel approach by using natural products, such as tocotrienols as proteasome modulators, which may lead to the development of new dietary supplements of tocotrienols for cardiovascular diseases, as well as others that are based on inflammation.

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Delta-tocotrienol protects mouse and human hematopoietic progenitors from gamma-irradiation through extracellular signal-regulated kinase/mammalian target of rapamycin signaling

Li XH, Fu D, Latif NH, Mullaney CP, Ney PH, Mog SR, Whitnall MH, Srinivasan V, Xiao M.

Haematologica. 2010 Dec;95(12):1996-2004.

Background: Exposure to γ-radiation causes rapid hematopoietic cell apoptosis and bone marrow suppression. However, there are no approved radiation countermeasures for the acute radiation syndrome. In this study, we demonstrated that natural δ-tocotrienol, one of the isomers of vitamin E, significantly enhanced survival in total body lethally irradiated mice. We explored the effects and mechanisms of δ-tocotrienol on hematopoietic progenitor cell survival after γ-irradiation in both in vivo and in vitro experiments.

Design And Methods: CD2F1 mice and human hematopoietic progenitor CD34(+) cells were treated with δ-tocotrienol or vehicle control 24 h before or 6 h after γ-irradiation. Effects of δ-tocotrienol on hematopoietic progenitor cell survival and regeneration were evaluated by clonogenicity studies, flow cytometry, and bone marrow histochemical staining. δ-tocotrienol and γ-irradiation-induced signal regulatory activities were assessed by immunofluorescence staining, immunoblotting and short-interfering RNA assay.

Results: δ-tocotrienol displayed significant radioprotective effects. A single injection of δ-tocotrienol protected 100% of CD2F1 mice from total body irradiation-induced death as measured by 30-day post-irradiation survival. δ-tocotrienol increased cell survival, and regeneration of hematopoietic microfoci and lineage(-)/Sca-1(+)/ckit(+) stem and progenitor cells in irradiated mouse bone marrow, and protected human CD34(+) cells from radiation-induced damage. δ-tocotrienol activated extracellular signal-related kinase 1/2 phosphorylation and significantly inhibited formation of DNA-damage marker γ-H2AX foci. In addition, δ-tocotrienol up-regulated mammalian target of rapamycin and phosphorylation of its downstream effector 4EBP-1. These alterations were associated with activation of mRNA translation regulator eIF4E and ribosomal protein S6, which is responsible for cell survival and growth. Inhibition of extracellular signal-related kinase 1/2 expression by short interfering RNA abrogated δ-tocotrienol-induced mammalian target of rapamycin phosphorylation and clonogenicity, and increased γ-H2AX foci formation in irradiated CD34(+) cells.

Conclusions: Our data indicate that δ-tocotrienol protects mouse bone marrow and human CD34(+) cells from radiation-induced damage through extracellular signal-related kinase activation-associated mammalian target of rapamycin survival pathways.

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Comparative hypoglycemic and nephroprotective effects of tocotrienol rich fraction (TRF) from palm oil and rice bran oil against hyperglycemia induced nephropathy in type 1 diabetic rats

Siddiqui S, Rashid Khan M, Siddiqui WA.

Chem Biol Interact. 2010 Dec 5;188(3):651-8.

Diabetic nephropathy (DN) is a serious complication confronted by patients with diabetes. Available data indicate that the development of DN is linked to hyperglycemia. Tocotrienol rich fraction (TRF) from palm oil (PO) and rice bran oil (RBO) has been shown to lower the blood glucose level in patients and preclinical animal models. This study was designed to investigate if TRF from PO and RBO could improve the renal function in DN by the virtue of their hypoglycemic and antioxidant activities. Male Wistar rats having an average body weight (bw) 250g were divided into four groups of six each .The first group served as diabetic control [injected with 55mg/kg bw of streptozotocin (STZ), intraperitoneally], while the second and third group received PO-TRF and RBO-TRF, respectively, by gavage at a dose of 200mg/kg bw/day, over a period of 8 weeks post-induction of diabetes. The fourth group comprised of age-matched male Wistar rats that received single intraperitoneal injection of normal saline only and served as control. After 8 weeks of STZ injection and TRF treatment, 24h urine was collected and animals were sacrificed. Fasting blood glucose, glycosylated hemoglobin, biochemical markers of renal function and oxidative stress were evaluated in serum, urine and kidney tissue. The results show that treatment with PO-TRF as well as RBO-TRF significantly improved the glycemic status and renal function in type 1 diabetic rats but PO-TRF afforded greater efficiency at similar dose as compared to RBO-TRF. In conclusion, PO-TRF was found to be more effective hypoglycemic and nephroprotective agent in DN than RBO-TRF.

d-δ-Tocotrienol-mediated cell cycle arrest and apoptosis in human melanoma cells

Fernandes NV, Guntipalli PK, Mo H.

Anticancer Res. 2010 Dec;30(12):4937-44.

Background: The rate-limiting enzyme of the mevalonate pathway, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, provides essential intermediates for the prenylation or dolichylation of growth-related proteins. d-δ-tocotrienol, a post-transcriptional down-regulator of HMG CoA reductase, suppresses the proliferation of murine B16 melanoma cells. Dietary d-δ-tocotrienol suppresses the growth of implanted B16 melanomas without toxicity to host mice.

Materials And Methods: The proliferation of human A2058 and A375 melanoma cells following a 72 h incubation in 96-well plates was measured by CellTiter 96® Aqueous One Solution. Cell cycle distribution was determined by flow cytometry. Fluorescence microscopy following acridine orange and ethidium bromide dual staining and procaspase-3 cleavage were used to detect apoptosis. Western-blot was employed to measure protein expression.

Results: d-δ-Tocotrienol induced dose-dependent suppression of cell proliferation with 50% inhibitory concentrations (IC(50)) of 37.5 ± 1.4 (A2058) and 22.3 ± 1.8 (A375) μmol/l, respectively (data are reported as mean ± standard deviation). d-δ-Tocotrienol-mediated cell cycle arrest at the G(1) phase was accompanied by reduced expression of cyclin-dependent kinase 4. Concomitantly, d-δ-tocotrienol induced caspase-3 activation and apoptosis. The impact of d-δ-tocotrienol on A2058 cell proliferation was potentiated by lovastatin (IC(50)=3.1 ± 0.5 μmol/l), a competitive inhibitor of HMG CoA reductase.

Conclusion: d-δ-Tocotrienol may have potential application in melanoma chemoprevention and/or therapy.