γ-Tocotrienol but not γ-tocopherol blocks STAT3 cell signaling pathway through induction of protein-tyrosine phosphatase SHP-1 and sensitizes tumor cells to chemotherapeutic agents

Kannappan R, Yadav VR, Aggarwal BB.

J Biol Chem. 2010 Oct 22;285(43):33520-8. Epub 2010 Aug 18.

Although γ-tocotrienol (T3), a vitamin E isolated primarily from palm and rice bran oil, has been linked with anticancer activities, the mechanism of this action is poorly understood. In this study, we investigated whether γ-T3 can modulate the STAT3 cell signaling pathway, closely linked toinflammation and tumorigenesis. We found that γ-T3 but not γ-tocopherol, the most common saturated form of vitamin E, inhibited constitutive activation of STAT3 in a dose- and time-dependent manner, and this inhibition was not cell type-specific. γ-T3 also inhibited STAT3 DNA binding. This correlated with inhibition of Src kinase and JAK1 and JAK2 kinases. Pervanadate reversed the γ-T3-induced down-regulation of STAT3 activation, suggesting the involvement of a protein-tyrosine phosphatase. When examined further, we found that γ-T3 induced the expression of the tyrosine phosphatase SHP-1, and gene silencing of the SHP-1 by small interfering RNA abolished the ability of γ-T3 to inhibit STAT3 activation, suggesting a vital role for SHP-1 in the action of γ-T3. Also γ-T3 down-modulated activation of STAT3 and induced SHP-1 in vivo. Eventually, γ-T3 down-regulated the expression of STAT3-regulated antiapoptotic (Bcl-2, Bcl-xL, and Mcl-1), proliferative (cyclin D1), and angiogenic (VEGF) gene products; and this correlated with suppression of proliferation, the accumulation of cells in sub-G(1) phase of the cell cycle, and induction of apoptosis. This vitamin also sensitized the tumor cells to the apoptotic effects of thalidomide and bortezomib. Overall, our results suggest that γ-T3 is a novel blocker of STAT3 activation pathway both in vitro and in vivo and thus may have potential in prevention and treatment of cancers.

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This study compared two dietary phytochemicals, grape-derived resveratrol and palm oil-derived γ-tocotrienol, either alone or in combination, on the contribution of autophagy in cardioprotection during ischaemia and reperfusion. Sprague-Dawley rats weighing between 250 and 300 g were randomly assigned to one of the following groups: vehicle, ischaemia/reperfusion (I/R), resveratrol + I/R, γ-tocotrienol + I/R, resveratrol +γ-tocotrienol + I/R. For resveratrol treatments, the rats were gavaged with resveratrol (2.5 mg/kg) for 15 days while for γ-tocotrienol experiments the rats were gavaged with γ-tocotrienol (0.3 mg/kg) for 30 days. For the combined resveratrol +γ-tocotrienol experiments, the rats were gavaged with γ-tocotrienol for 15 days, and then gavaging continued with resveratrol along with γ-tocotrienol for a further period of 15 days. After 30 days, isolated perfused hearts were subjected to 30 min. of global ischaemia followed by 2 hrs of reperfusion. Our results showed for the first time that at least in part, the cardioprotection (evidenced from the ventricular performance, myocardial infarct size and cardiomyocyte apoptosis) with resveratrol and γ-toctrienol was achieved by their abilities to induce autophagy. Most importantly, resveratrol and γ-tocotrienol acted synergistically providing greater degree of cardioprotection simultaneously generating greater amount of survival signal through the activation of Akt-Bcl-2 survival pathway. Autophagy was accompanied by the activation of Beclin and LC3-II as well as mTOR signalling, which were inhibited by either 3-methyl adenine (3-MA) or Wortmannin. The autophagy was confirmed from the results of transmission electron microscopy and light microscopy as well as with confocal microscopy. It is tempting to speculate that during ischaemia and reperfusion autophagy along with enhanced survival signals helps to recover the cells from injury.

A tocotrienol-rich fraction from grape seeds inhibits oxidative stress induced by tert-butyl hydroperoxide in HepG2 cells

Choi Y, Lee SM, Kim Y, Yoon J, Jeong HS, Lee J.

J Med Food. 2010 Oct;13(5):1240-6.

We evaluated the protective effect of a tocotrienol-rich fraction (TRF) from grape seeds on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells. Generation of cellular reactive oxygen species (ROS), concentrations of cellular lipid peroxidation products and reduced glutathione, and antioxidant enzyme activity were used as biomarkers of cellular oxidative status. Cells pretreated with TRF (10–500 μg/mL) showed an increased resistance to oxidative stress in a dose-dependent manner, as revealed by a higher percentage of surviving cells compared to control cells. Pretreatment with TRF (5–100 μg/mL) prevented the decrease in reduced glutathione and the increase in malondialdehyde and ROS evoked by TBHP in HepG2 cells. Moreover, TRF pretreatment prevented a significant increase in glutathione peroxidase, catalase, and superoxide dismutase activities induced by TBHP. These results show that TRF has significant protective ability against TBHP-induced oxidative insult and that the modulation of antioxidant enzymes by TRF may have an important antioxidant effect on HepG2 cells.

Effectiveness of tocotrienol-rich fraction combined with tamoxifen in the management of women with early breast cancer: A pilot clinical trial

Nesaretnam K, Selvaduray KR, Abdul Razak G, Veerasenan SD, Gomez PA.

Breast Cancer Res. 2010;12(5):R81. Epub 2010 Oct 8.

INTRODUCTION: Basic research has indicated that tocotrienols have potent antiproliferative and proapoptotic effects that would be expected to reduce the effect of breast cancer.

METHODS: We conducted a double-blinded, placebo-controlled pilot trial to test the effectiveness of adjuvant tocotrienol therapy in combination with tamoxifen for five years in women with early breast cancer. Two-hundred-forty women, aged between 40-60 years, with either tumor node metastases (TNM) Stage I or II breast cancer and estrogen receptor (ER) positive tumors were non-randomly assigned to two groups. The intervention group received tocotrienol rich fraction (TRF) plus tamoxifen whilst the control group received placebo plus tamoxifen, for five years.

RESULTS: During the five years of study, 8 patients died due to breast cancer while 36 patients developed local or systemic recurrence. Five-year breast cancer specific survival was 98.3% (95% confidence interval (CI): 95.9% to 100%) in the intervention group and 95%, (95% CI: 91.1% to 98.9%) in the control group, while 5-years disease free survival was 86.7% (95% CI: 80.6% to 92.8%) and 83.3% (95% CI: 76.6% to 90.0%), respectively. Risk of mortality due to breast cancer was 60% (HR: 0.40; 95% CI: 0.08 to 2.05) lower in the intervention group versus the controls following adjustment for age, ethnicity, stage and lymph node status but this was not statistically significant. Adjuvant TRF therapy was not associated with breast cancer recurrence (HR: 0.84; 95% CI: 0.43-1.65).

CONCLUSIONS:From the current study, there seems to be no association between adjuvant tocotrienol therapy and breast cancer specific survival in women with early breast cancer.

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Gamma delta tocotrienols reduce hepatic triglyceride synthesis and VLDL secretion

Zaiden N, Yap WN, Ong S, Xu CH, Teo VH, Chang CP, Zhang XW, Nesaretnam K, Shiba S, Yap YL.

J Atheroscler Thromb. 2010 Oct 27;17(10):1019-32.

Aim: Present study aimed to elucidate the suppression of serum lipids by gamma- and delta-tocotrienol (γδT3).

Methods: The lipid-lowering effects of γδT3 were investigated using HepG2 liver cell line, hypercholesterolemic mice and borderline-high cholesterol patients.

Results: In-vitro results demonstrated two modes of action. First, γδT3 suppressed the upstream regulators of lipid homeostasis genes (DGAT2, APOB100, SREBP1/2 and HMGCR) leading to the suppression of triglycerides, cholesterol and VLDL biosyntheses. Second, γδT3 enhanced LDL efflux through induction of LDL receptor (LDLr) expression. Treatment of LDLr-deficient mice with 1 mg/day (50 mg/kg/day) γδT3 for one-month showed 28%, 19% reduction in cholesterol and triglyceride levels respectively, whereas HDL level was unaltered. The lipid-lowering effects were not affected by alpha-tocopherol (αTP). In a placebo-controlled human trial using 120 mg/day γδT3, only serum triglycerides were lowered by 28% followed by concomitant reduction in the triglyceride-rich VLDL and chylomicrons. In contrast, total cholesterol, LDL and HDL remained unchanged in treated and placebo groups. The discrepancies between in-vitro, in-vivo and human studies may be attributed to the differential rates of post-absorptive γδT3 degradation and LDL metabolism.

Conclusion: Reduction in triglycerides synthesis and transport may be the primary benefit caused by ingesting γδT3 in human.

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Differential suppression of proliferation in MCF-7 and MDA-MB-231 breast cancer cells exposed to alpha-, gamma- and delta-tocotrienols is accompanied by altered expression of oxidative stress modulatory enzymes

Hsieh TC, Elangovan S, Wu JM.

Anticancer Res. 2010 Oct;30(10):4169-76.

Tocotrienols belong to the vitamin E family of chemicals known to have potent anti-proliferative and apoptotic activities against a variety of cancer cells with little to no comparable influence on the normal cells. Whether tocotrienols control the expression of phase II antioxidant enzymes in the context of their anti-carcinogenic mechanisms has not been investigated. The present studies were performed to test whether the differential growth inhibition resulting from exposure to α-, γ- and δ-tocotrienols in estrogen receptor-positive human MCF-7 and estrogen receptor-negative MDA-MB-231 breast cancer cells might be accompanied by changes in phase II antioxidant enzymes. Cell proliferation and clonogenicity in both cell lines were significantly inhibited by γ- and δ-tocotrienols with little affect when cells were similarly exposed to α-tocotrienol, at doses up to 10 μM. The expression and activity of several antioxidant enzymes in 10 μM tocotrienol-treated cells were determined by Western blot and biochemical assays. In MDA-MB-231 cells, δ- was more active than α- or γ-tocotrienols in up-regulating glutathione peroxidase; however, the three tocotrienols had comparable activity in inducing thioredoxin. In MCF-7 cells, expression of quinone reductase 2 and thioredoxin was increased by γ- and δ-tocotrienols, whereas quinone reductase 1 was unaffected by exposure to the tocotrienols. The tocotrienols also did not affect the expression and activity of superoxide dismutase in both MCF-7 and MDA-MB-231 cells, but increased catalase activity concomitant with slight reduction in the catalase expression. In MDA-MB-231 cells, treatment by tocotrienols led to several fold increase of NRF2 expression marked by corresponding decrease in KEAP1 levels. By contrast, no significant change in NRF2 and KEAP1 levels was observed in MCF-7 cells. These studies demonstrate that different tocotrienols show distinct and selective activity in regulating the NRF2-KEAP1, in coordination with the induced expression of cytoprotective oxidative stress modulatory genes and regulation of proliferation in breast cancer cells.

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