Mechanisms Mediating the Synergistic Anticancer Effects of Combined γ-Tocotrienol and Celecoxib Treatment

Shirode AB, Sylvester PW.

J Bioanal Biomed. 2011 Jan 10;3:1-7.

Aim: To characterize the intracellular signaling mechanisms mediating the synergistic anticancer effects of combined γ-tocotrienol and celecoxibtreatment in neoplastic +SA mouse mammary epithelial cells in vitro.

Methods: +SA mammary tumor cells in different treatment groups were maintained in serum-free defined media containing 10ng/ml EGF as a mitogen and exposed to various doses of γ-tocotrienol and celecoxib alone or in combination. After a 96 hr culture period, cells were collected and whole cell lysates were subjected to Western blot analysis to determine treatment effects on intracellular signaling proteins associated with EGF-dependent mitogenesis and survival, and prostaglandin synthesis and responsiveness.

Results: Treatment with high doses of γ-tocotrienol or celecoxib alone inhibited Akt activation and downstream signaling and NFκB activation. Similar treatment with γ-tocotrienol also decreased concentration and activation of ErbB2-4 receptors, whereas celecoxib only inhibited ErbB2-4 receptor activation. In contrast, combined treatment with subeffective doses of γ-tocotrienol and celecoxib resulted in a large decrease ErbB2-4 receptor levels and activation, a decrease in PGE(2) levels, and a corresponding increase in prostaglandin EP2 and EP4 receptor levels. Combinedtreatment also induced an increase in the prostaglandin catabolizing enzyme, PGDH.

Conclusion: The synergistic anticancer effects of combined low dose γ-tocotrienol and celecoxib treatment in +SA mammary tumor cells are mediated by COX-2-dependent mechanisms associated with a suppression in PGE(2) levels, as well as, COX-2-independent mechanisms associated with a reduction in ErbB2-4 receptor levels, activation, and subsequent reduction in downstream Akt and NFκB mitogenic signaling.

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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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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.

Tocotrienols, the vitamin E of the 21st century: Its potential against cancer and other chronic diseases

Aggarwal BB, Ahn KS, Sundaram C, Prasad S, Kannappan R.

Biochem Pharmacol, 2010;80(11):1613-31

Initially discovered in 1938 as a “fertility factor,” vitamin E now refers to eight different isoforms that belong to two categories, four saturated analogues (alpha, beta, gamma, and delta) called tocopherols and four unsaturated analogues referred to as tocotrienols. While the tocopherols have been investigated extensively, little is known about the tocotrienols. Very limited studies suggest that both the molecular and therapeutic targets of the tocotrienols are distinct from those of the tocopherols. For instance, suppression of inflammatory transcription factor NF-kappaB, which is closely linked to tumorigenesis and inhibition of HMG-CoA reductase, mammalian DNA polymerases and certain protein tyrosine kinases, is unique to the tocotrienols. This review examines in detail the molecular targets of the tocotrienols and their roles in cancer, bone resorption, diabetes, and cardiovascular and neurological diseases at both preclinical and clinical levels. As disappointment with the therapeutic value of the tocopherols grows, the potential of these novel vitamin E analogues awaits further investigation.

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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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Regulation of survival, proliferation, invasion, angiogenesis, and metastasis of tumor cells through modulation of inflammatory pathways by nutraceuticals

Gupta SC, Kim JH, Prasad S, Aggarwal BB.

Cancer Metastasis Rev. 2010 Sep;29(3):405-34.

Almost 25 centuries ago, Hippocrates, the father of medicine, proclaimed “Let food be thy medicine and medicine be thy food.” Exploring the association between diet and health continues today. For example, we now know that as many as 35% of all cancers can be prevented by dietary changes. Carcinogenesis is a multistep process involving the transformation, survival, proliferation, invasion, angiogenesis, and metastasis of the tumor and may take up to 30 years. The pathways associated with this process have been linked to chronic inflammation, a major mediator of tumor progression. The human body consists of about 13 trillion cells, almost all of which are turned over within 100 days, indicating that 70,000 cells undergo apoptosis every minute. Thus, apoptosis/cell death is a normal physiological process, and it is rare that a lack of apoptosis kills the patient. Almost 90% of all deaths due to cancer are linked to metastasis of the tumor. How our diet can prevent cancer is the focus of this review. Specifically, we will discuss how nutraceuticals, such as allicin, apigenin, berberine, butein, caffeic acid, capsaicin, catechin gallate, celastrol, curcumin, epigallocatechin gallate, fisetin, flavopiridol, gambogic acid, genistein, plumbagin, quercetin, resveratrol, sanguinarine, silibinin, sulforaphane, taxol, gamma-tocotrienol, and zerumbone, derived from spices, legumes, fruits, nuts, and vegetables, can modulate inflammatory pathways and thus affect the survival, proliferation, invasion, angiogenesis, and metastasis of the tumor. Various cell signaling pathways that are modulated by these agents will also be discussed.

The tocotrienol-rich fraction from rice bran enhances cisplatin-induced cytotoxicity in human mesothelioma H28 cells.

Nakashima K, Virgona N, Miyazawa M, Watanabe T, Yano T.

Phytother Res. 2010 Sep;24(9):1317-21.

Resistance to chemotherapy (chemoresistance) is a serious problem in malignant mesothelioma, a highly aggressive neoplasm. Gamma-tocotrienol (gamma-T3) can sensitize various cancerous cells to chemotherapeutic agents by inhibiting pathways that lead to treatment resistance. In this study, we investigated the modulating effect of tocotrienol-rich fraction (TRF) from rice bran, which is abundant in gamma-T3, on chemoresistance in human MM H28 cells. TRF treatment caused a marked reduction in the viability of H28 cells in a dose-dependent manner, while cisplatin treatment had no effect on the cells, indicating that H28 cells are resistant to cisplatin. A significant increase in cytotoxicity was observed in H28 cells treated with TRF, and this effect was enhanced by the combination treatment with cisplatin. The cytotoxic effect was closely related to the inhibition of phosphatidylinositol 3-kinase (PI3K)-AKT signaling. Inactivation of Akt signaling by TRF or the combination with cisplatin mitigated cisplatin-induced activation of Akt, resulting in reducing the chemoresistance H28 cells to cisplatin. Reduced cell viability and attenuated chemoresistance of the H28 cells against cisplatin were also observed following the use of a PI3K inhibitor, LY294002. These results suggest that the combination therapy of cisplatin with TRF is a plausible strategy for achieving tolerance for the chemotherapeutic agent in MM therapy.

Effect of δ-tocotrienol on melanin content and enzymes for melanin synthesis in mouse melanoma cells

Michihara A, Ogawa S, Kamizaki Y, Akasaki K.

Biol Pharm Bull. 2010;33(9):1471-6.

In the present study, we investigated the dose-dependent effect of delta-tocotrienol long term (48, 72 h) on the melanin content of cells treated with delta-tocotrienol, and whether cells treated with delta-tocotrienol for long a time show cytotoxicity. We also examined whether other enzymes responsible for melanin biosynthesis, tyrosinase-related protein-1 (TRP-1) and -2 (TRP-2), are involved in the decrease in melanin levels. Protein levels in cells treated with 25 or 50 microM delta-tocotrienol for 48 h or 72 h were similar to those in control cells. Melanin content decreased by 44 (25 microM delta-tocotrienol) to 50% (50 microM) at 48 h, and by 14 to 21% at 72 h, compared to control levels. Tyrosinase activity, amounts of tyrosinase and TRP-1 decreased dependent on dose : by 50 (25 microM delta-tocotrienol) to 75% (50 microM), 20 to 45% and 42 to 82% at 48 h, and by 25 to 50%, 75 to 80% and 78 to 77% at 72 h, respectively. Although the amount of TRP-2 increased by 20% on treatment with 25 microM delta-tocotrienol for 48 h, it decreased by 52% on treatment with 50 microM delta-tocotrienol for 48 h. The amount of TRP-2 dose-dependently decreased by 55% and 75% on 72 h by treatment with 25 and 50 microM delta-tocotrienol, respectively. From these findings, delta-tocotrienol at up to 50 microM dose-dependently caused a reduction in melanin content by the decrease of TRP-1 and TRP-2 as well as tyrosinase, and no cytotoxicity.

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Gamma-tocotrienol promotes TRAIL-induced apoptosis through reactive oxygen species/extracellular signal-regulated kinase/p53-mediated upregulation of death receptors

Kannappan R, Ravindran J, Prasad S, Sung B, Yadav VR, Reuter S, Chaturvedi MM, Aggarwal BB.

Mol Cancer Ther. 2010 Aug;9(8):2196-207. Epub 2010 Aug 3.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor superfamily, is in clinical trials for cancer therapy, but its anticancer potential is limited by the development of resistance. We investigated the ability of tocotrienol (T3), an unsaturated vitamin E present in palm oil, rice bran, barley, oats, and wheat germ, to sensitize tumor cells to TRAIL. Results from esterase staining, colony formation, caspase activation, and sub-G(1) cell cycle arrest revealed that gamma-T3 can sensitize human colon cancer cells to TRAIL. When examined for the mechanism, we found that gamma-T3 significantly downregulated the expression of antiapoptotic proteins (c-IAP2 and Bcl-xL). We also found that gamma-T3, but not tocopherol, induced the expression of the TRAIL receptors death receptor (DR)-4 and DR5. This induction was not cell type specific, as upregulation was also found in pancreatic, kidney, and leukemic cells. Upregulation of DRs by gamma-T3 required the production of reactive oxygen species (ROS), and sequestering of ROS abolished both upregulation of the receptors and potentiation of TRAIL-induced apoptosis. Induction of DRs by gamma-T3 also required activation of extracellular signal-regulated kinase 1 (ERK1), as silencing of ERK1 by specific siRNA abrogated the upregulation of TRAIL receptors. Further, induction of DRs by gamma-T3 required the expression of p53 and Bax, as no induction of the receptors was found in colon cancer cells with deletion of these genes. Overall, our results show that gamma-T3 sensitizes tumor cells to TRAIL by upregulating DRs through the ROS/ERK/p53 pathway and by downregulating cell survival proteins.

Gamma-Tocotrienol modulates the paracrine secretion of VEGF induced by cobalt(II) chloride via ERK signaling pathway in gastric adenocarcinoma SGC-7901 cell line.

Bi S, Liu JR, Li Y, Wang Q, Liu HK, Yan YG, Chen BQ, Sun WG.

Toxicology. 2010 Jul;274(1-3):27-33.

Hypoxia is a common characteristic feature of solid tumors, and carcinoma cells are known to secrete many growth factors. These growth factors, such as vascular endothelial growth factor (VEGF), play a major role in the regulation of tumor angiogenesis and metastasis. In this study, the effect of gamma-tocotrienol, a natural product commonly found in palm oil and rice bran, on the accumulation of HIF-1alpha protein and the paracrine secretion of VEGF in human gastric adenocarcinoma SGC-7901 cell line induced by cobalt(II) chloride (as a hypoxia mimic) was investigated. These results showed that cobalt(II) chloride induced the high expression of VEGF in SGC-7901 cells at dose of 150 micromol/L for 24h. Both basal level and cobalt(II) chloride-induced HIF-1alpha protein accumulation and VEGF paracrine secretion were inhibited in SGC-7901 cells treated with gamma-tocotrienol at 60 micromol/L treatment for 24 h. U0126, a MEK1/2 inhibitor, decreased the expression of HIF-1alpha protein and the paracrine secretion of VEGF under normoxic and hypoxic conditions. In this study, gamma-tocotrienol also significantly inhibited the hypoxia-stimulated expression of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2). The mechanism seems to involve in inhibiting hypoxia-mediated activation of p-ERK1/2, it leads to a marked decrease in hypoxia-induced HIF-1alpha protein accumulation and VEGF secretion. These data suggest that HIF-1alpha/VEGF could be a promising target for gamma-tocotrienol in an effective method of chemoprevention and chemotherapy in human gastric cancer.