α-Tocopherol as functional excipient for resveratrol and coenzyme Q10-loaded SNEDDS for improved bioavailability and prophylaxis of breast cancer.

Jain S, Garg T, Kushwah V, Thanki K, Agrawal AK, Dora CP.

J Drug Target. 2017 Mar 8:1-12. doi: 10.1080/1061186X.2017.1298603. [Epub ahead of print]

Abstract

The present study evaluates the prophylactic efficacy of α-tocopherol (α-TOH), resveratrol (RES), and coenzyme Q10 (CoQ10) co-loaded self-nanoemulsifying drug delivery system (α-TOH-RES-CoQ10 SNEDDS) in 7,12-Dimethylbenz[a]anthracene (DMBA) induced breast cancer model. SNEDDS formulation components were rationally selected and optimized for maximum drug loading by applying the design of experiments and further evaluated for stability in simulated gastrointestinal fluids, functional stability of antioxidants, in vitro release, Caco-2 cell uptake, oral bioavailability and prophylactic anticancer activity. The SNEDDS demonstrated excellent stability in stimulated gastrointestinal fluids. The functional activity of antioxidants was confirmed by 2,2-diphenylpicrylhydrazyl (DPPH) scavenging assay wherein significantly (p > .05) higher antioxidant activity was observed in case of SNEDDS as compared with free antioxidants. Coumarin 6 (C-6)-loaded SNEDDS formulation demonstrated remarkably higher Caco-2 cell uptake in comparison with free C-6, indicative of efficient internalization of sub-micron SNEDDS droplets by Caco-2 cells. In line with Caco-2 cell uptake observations, α-TOH-RES-CoQ10-SNEDDS showed ∼2.30- and ∼3.64-fold increase in the AUC0-∞ values of RES and CoQ10 in comparison with free antioxidants. Significantly lower (p < .001) tumor volume (∼327 mm3) was found in case of animals treated with α-TOH-RES-CoQ10-SNEDDS in comparison with free antioxidant combination (∼1070 mm3) and DMBA control (∼1540 mm3) groups. Conclusively, the proposed strategy posed great potential in improving the prophylactic activity of antioxidants and hold promise for further exploration.

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Inhibitory effects of γ- and δ-tocopherols on estrogen-stimulated breast cancer in vitro and in vivo.

Bak MJ, Das Gupta S, Wahler J, Lee HJ, Li X, Lee MJ, Yang CS, Suh N.

Cancer Prev Res (Phila). 2017 Jan 17. pii: canprevres.0223.2016. doi: 10.1158/1940-6207.CAPR-16-0223. [Epub ahead of print]

Abstract

Estrogens have been implicated as complete carcinogens for breast and other tissues through mechanisms involving increased cell proliferation, oxidative stress and DNA damage. Because of their potent antioxidant activity and other effects, tocopherols have been shown to exert anti-tumor activities in various cancers. However, limited information is available on the effect of different forms of tocopherols in estrogen-mediated breast cancer. To address this, we examined the effects of α-, γ- and δ-tocopherols as well as a natural γ-tocopherol rich mixture of tocopherols, γ-TmT, on estrogen-stimulated MCF-7 cells in vitro and in vivo. For the in vivo studies, MCF-7 cells were injected into the mammary fat pad of immunodeficient mice previously implanted with estrogen pellets. Mice were then administered diets containing 0.2% α-, γ-, δ-tocopherol or γ-TmT for 5 weeks. Treatment with α-, γ-, δ-tocopherols and γ-TmT reduced tumor volumes by 29% (p<0.05), 45% (p<0.05), 41% (p<0.05) and 58% (p<0.01), as well as tumor weights by 20%, 37% (p<0.05), 39% (p<0.05) and 52% (p<0.05), respectively. γ- and δ-Tocopherols and γ-TmT inhibited the expression of cell proliferation-related genes such as cyclin D1 and c-Myc, and estrogen-related genes such as TFF/pS2, cathepsin D and progesterone receptor in estrogen-stimulated MCF-7 cells in vitro. Further, γ- and δ-tocopherols decreased the levels of estrogen-induced oxidative stress and nitrosative stress markers, 8-hydroxy-2′-deoxyguanosine and nitrotyrosine, as well as the DNA damage marker, γ-H2AX. Our results suggest that γ- and δ-tocopherols and the γ-tocopherol rich mixture are effective natural agents for the prevention and treatment of estrogen-mediated breast cancer.

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Role of Rac1/WAVE2 Signaling in Mediating the Inhibitory Effects of γ-Tocotrienol on Mammary Cancer Cell Migration and Invasion.

Algayadh IG, Dronamraju V, Sylvester PW.

Biol Pharm Bull. 2016;39(12):1974-1982.

Abstract

The majority of breast cancer deaths result from the progression of this disease to a metastatic phenotype. Rac1 and Cdc42 are Rho family members that together with their downstream effectors, Wiskott-Aldrich Syndrome protein-family verprolin-homologous protein 2 (WAVE2) and Arp2/3, play an important role in cytoskeletal reorganization and the formation of membrane protrusions that promote cancer cell migration and invasion. γ-Tocotrienol, is a natural isoform within the vitamin E family of compounds that inhibits breast cancer cell growth and progression by suppressing various signaling pathways involved in mitogenic signaling and metastatic progression. Studies were conducted to examine the effects of γ-tocotrienol on Rac1/WAVE2 signaling dependent migration and invasion in highly metastatic mouse +SA and human MDA-MB-231 mammary cancer cells. Exposure to γ-tocotrienol resulted in a dose-responsive decrease in Rac1/WAVE2 signaling as characterized by a suppression in the levels of Rac1/Cdc42, phospho-Rac1/Cdc42, WAVE2, Arp2, and Arp3 expression. Additional studies also demonstrated that similar treatment with γ-tocotrienol resulted in a significant reduction in tumor cell migration and invasion. Taken together, these findings indicate that γ-tocotrienol treatment effectively inhibits Rac1/WAVE2 signaling and reduces metastatic phenotypic expression in mammary cancer cells, suggesting that γ-tocotrienol may provide some benefit as a novel therapeutic approach in the treatment of metastatic breast cancer.

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Gamma-tocotrienol reverses multidrug resistance of breast cancer cells with a mechanism distinct from that of atorvastatin.

Ding Y, Peng Y, Deng L, Fan J, Huang B.

J Steroid Biochem Mol Biol. 2016 Nov 15. pii: S0960-0760(16)30323-5. doi: 10.1016/j.jsbmb.2016.11.009. [Epub ahead of print]

Abstract

In addition to its antioxidant properties, γ-tocotrienol also has the ability to inhibit HMG-CoA reductase, which is the key enzyme in the mevalonate pathway for cholesterol biosynthesis. Statins, the competitive inhibitors of HMG-CoA reductase, display potent anticancer activity and reversal ability of multidrug resistance in a variety of tumor cells, which is believed to be due to their inhibition of HMG-CoA reductase. Here, we determined the role of the mevalonate pathway in γ-tocotrienol-mediated reversal of multidrug resistance in cancer cells. We found both γ-tocotrienol and atorvastatin effectively reversed multidrug resistance of MCF-7/Adr and markedly inhibited the intracellular levels of FPP and GGPP. Exogenous addition of mevalonate or FPP and GGPP almost completely prevented the reversal ability of atorvastatin but only partly attenuated the reversal effect of γ-tocotrienol on doxorubicin resistance. In addition, γ-tocotrienol actively inhibited the expression of P-gp and increased the accumulation of doxorubicin in cells, which led to the enhanced G2/M arrest and cell apoptosis. Taken together, γ-tocotrienol reversed the multidrug resistance of MCF-7/Adr with a mechanism distinct from that of atorvastatin. Instead of the mevalonate pathway, the inhibition of P-gp expression is a potential mechanism by which γ-tocotrienol reverses multidrug resistance in MCF-7/Adr.

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Tocotrienol Nanoemulsion Platform of Curcumin Elicit Elevated Apoptosis and Augmentation of Anticancer Efficacy against Breast and Ovarian Carcinomas.

Steuber N, Vo K, Wadhwa R, Birch J, Iacoban P, Chavez P, Elbayoumi TA.

Int J Mol Sci. 2016 Oct 26;17(11). pii: E1792.

Abstract

Vitamin E (VE) tocotrienols (T3), recognized for their cancer-specific anti-proliferative and pro-apoptotic activities, have been previously fabricated into bio-active nanoemulsion (NE) formulations. Here, our viscosity-adapted δ-T3 NE platform was developed to additionally incorporate curcumin (CUR), which is known for its potent suppression of signaling pathways involved in malignant cell growth, survival and metastasis. Thanks to efficient 70:30 wt % surfactant mix of Lutrol F-127:VE-TPGS, in conjunction with optimal CUR loading, a prototype CUR in δ-T3 NE was successfully prepared. Model CUR/δ-T3 NE demonstrated excellent nano-scale aspects (mean particle size = 261 nm, PDI = 0.27, and ζ-potential = -35 mV), pharmaceutical stability, and controlled release properties. Suitability for systemic administration was also verified via standardized in vitro biocompatibility and hemocompatibility assays. In two human cancer cells (MCF-7 and OVCAR-8), our CUR/δ-T3 NE prominently suppressed constitutive NF-κB activation, and significantly induced apoptosis. Finally, the combined CUR/δ-T3 NE produced superior cytotoxicity profiles, in concentration- and time-dependent manners (p ≤ 0.05), at least three to four folds lower IC50 than in closest CUR control. The strong synergism, estimated in both cultured carcinomas, revealed the augmented therapeutic efficacy of our CUR/δ-T3 NE combined platform, supporting its strong potential towards pharmaceutical development for cancer therapy.

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γ-Tocotrienol reversal of epithelial-to-mesenchymal transition in human breast cancer cells is associated with inhibition of canonical Wnt signalling.

Ahmed RA, Alawin OA, Sylvester PW.

Cell Prolif. 2016 Aug;49(4):460-70. doi: 10.1111/cpr.12270.

Abstract

Frizzled-7 (FZD7) receptor-dependent activation of the canonical Wnt/β-catenin pathway plays a crucial role in epithelial-to-mesenchymal transition (EMT) and breast cancer metastasis. FZD7 and its co-receptor, low-density lipoprotein receptor-related protein 6 (LRP6), are highly expressed in MDA-MB-231 and T-47D breast cancer cells, and endogenous ligands for FZD7 include Wnt3a and Wnt5a/b. γ-Tocotrienol, a natural isoform of vitamin E, inhibits human breast cancer cell proliferation and EMT. Here, studies have been conducted to investigate the role of the canonical Wnt pathway in mediating inhibitory effects of γ-tocotrienolon EMT in human breast cancer cells. Results show γ-Tocotrienol was found to induce dose-responsive inhibition of MDA-MB-231 and T-47D cell growth at doses that had no effect on immortalized normal MCF-10A mammary epithelial cells. These growth inhibitory effects were associated with suppression in canonical Wnt signalling, reversal of EMT and significant reduction in breast cancer cell motility. In conclusion, γ-Tocotrienol suppression of metastatic breast cancer cell proliferation and EMT was associated with suppression of the canonical Wnt/β-catenin signalling pathway.

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Elimination of ALDH+ breast tumor initiating cells by docosahexanoic acid and/or gamma tocotrienol through SHP-1 inhibition of Stat3 signaling.

Xiong A, Yu W, Liu Y, Sanders BG, Kline K.

Mol Carcinog. 2016 May;55(5):420-30. doi: 10.1002/mc.22291.

Abstract

Study investigated the ability of docosahexaenoic acid (DHA) alone and in combination with gamma-tocotrienol (γT3) to eliminate aldehyde dehydrogenase positive (ALDH+) cells and to inhibit mammosphere formation, biomarker and functional assay for tumor initiating cells (TICs), respectively, in human triple negative breast cancer cells (TNBCs), and investigated possible mechanisms of action. DHA upregulated Src homology region 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) protein levels and suppressed levels of phosphorylated signal transducer and activator of transcription-3 (pStat3) and its downstream mediators c-Myc, and cyclin D1. siRNA to SHP-1 enhanced the percentage of ALDH+ cells and Stat-3 signaling, as well as inhibited, in part, the ability of DHA to reduce the percentage of ALDH+ cells and Stat-3 signaling. γT3 alone and in combination with DHA reduced ALDH+ TNBCs, up-regulated SHP-1 protein levels, and suppressed Stat-3 signaling. Taken together, data demonstrate the anti-TIC potential of achievable concentrations of DHA alone as well as in combination with γT3.

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Molecular and Biochemical Analysis of the Estrogenic and Proliferative Properties of Vitamin E Compounds.

Khallouki F, de Medina P, Caze-Subra S, Bystricky K, Balaguer P, Poirot M, Silvente-Poirot S.

Front Oncol. 2016 Jan 5;5:287. doi: 10.3389/fonc.2015.00287.

Abstract

Tocols are vitamin E compounds that include tocopherols (TPs) and tocotrienols (TTs). These lipophilic compounds are phenolic antioxidants and are reportedly able to modulate estrogen receptor β (ERβ). We investigated the molecular determinants that control their estrogenicity and effects on the proliferation of breast cancer cells. Docking experiments highlighted the importance of the tocol phenolic groups for their interaction with the ERs. Binding experiments confirmed that they directly interact with both ERα and ERβ with their isoforms showing potencies in the following order: δ-tocols > γ-tocols > α-tocols. We also found that tocols activated the transcription of an estrogen-responsive reporter gene that had been stably transfected into cells expressing either ERα or ERβ. The role of the phenolic group in tocol-ER interaction was further established using δ-tocopherylquinone, the oxidized form of δ-TP, which had no ER affinity and did not induce ER-dependent transcriptional modulation. Tocol activity also required the AF1 transactivation domain of ER. We found that both δ-TP and δ-TT stimulated the expression of endogenous ER-dependent genes. However, whereas δ-TP induced the proliferation of ER-positive breast cancer cells but not ER-negative breast cancer cells, δ-TT inhibited the proliferation of both ER-positive and ER-negative breast cancer cells. These effects of δ-TT were found to act through the down regulation of HMG-CoA reductase (HMGR) activity, establishing that ERs are not involved in this effect. Altogether, these data show that the reduced form of δ-TP has estrogenic properties which are lost when it is oxidized, highlighting the importance of the redox status in its estrogenicity. Moreover, we have shown that δ-TT has antiproliferative effects on breast cancer cells independently of their ER status through the inhibition of HMGR. These data clearly show that TPs can be discriminated from TTs according to their structure.

Antiproliferative effects of γ-tocotrienol are associated with lipid raft disruption in HER2-positive human breast cancer cells.

Alawin OA, Ahmed RA, Ibrahim BA, Briski KP, Sylvester PW.

J Nutr Biochem. 2016 Jan;27:266-77. doi: 10.1016/j.jnutbio.2015.09.018.

Abstract

A large percentage of human breast cancers are characterized by excessive or aberrant HER2 activity. Lipid rafts are specialized microdomains within the plasma membrane that are required for HER2 activation and signal transduction. Since the anticancer activity of γ-tocotrienol is associated with suppression in HER2 signaling, studies were conducted to examine the effects of γ-tocotrienol on HER2 activation within the lipid raft microdomain in HER2-positive SKBR3 and BT474 human breast cancer cells. Treatment with 0-5μM γ-tocotrienol induced a significant dose-dependent inhibition in cancer cell growth after a 5-day culture period, and these growth inhibitory effects were associated with a reduction in HER2 dimerization and phosphorylation (activation). Phosphorylated HER2 was found to be primarily located in the lipid raft microdomain of the plasma membrane in vehicle-treated control groups, whereas γ-tocotrienol treatment significantly inhibited this effect. Assay of plasma membrane subcellular fractions showed that γ-tocotrienol also accumulates exclusively within the lipid raft microdomain. Hydroxypropyl-β-cyclodextrin (HPβCD) is an agent that disrupts lipid raft integrity. Acute exposure to 3mM HPβCD alone had no effect, whereas an acute 24-h exposure to 20μM γ-tocotrienol alone significantly decreased SKBR3 and BT474 cell viability. However, combined treatment with these agents greatly reduced γ-tocotrienolaccumulation in the lipid raft microdomain and cytotoxicity. In summary, these findings demonstrate that the anticancer effects of γ-tocotrienol are associated with its accumulation in the lipid raft microdomain and subsequent interference with HER2 dimerization and activation in SKBR3 and BT474 human breast cancer cells.

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Vitamin E therapy beyond cancer: tocopherol versus tocotrienol.

Peh HY, Daniel Tan WS, Liao W, Fred Wong WS.

Pharmacol Ther. 2015 Dec 16. pii: S0163-7258(15)00229-6

Abstract

The discovery of vitamin E (α-tocopherol) began in 1922 as a vital component required in reproduction. Today, there are eight naturally occurring vitamin E isoforms, namely α-, β-, γ- and δ-tocopherol and α-, β-, γ- and δ-tocotrienol. Vitamin E are potent antioxidants, capable of neutralizing free radicals directly by donating hydrogen from its chromanol ring. α-Tocopherol is regarded the dominant form in vitamin E as the α-tocopherol transfer protein in the liver binds mainly α-tocopherol, thus preventing its degradation. That contributed to the oversight of tocotrienols and resulted in less than 3% of all vitamin E publications studying tocotrienols. Nevertheless, tocotrienols have been shown to possess superior antioxidant and anti-inflammatory properties over α-tocopherol. In particular, inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase to lower cholesterol, attenuating inflammation via downregulation of transcription factor NF-κB activation, and potent radioprotectant against radiation damage are some properties unique to tocotrienols, not tocopherols. Aside from cancer, vitamin E has also been shown protective in bone, cardiovascular, eye, nephrological and neurological diseases. In light of the different pharmacological properties of tocopherols and tocotrienols, it becomes critical to specify which vitamin E isoform(s) are being studied in any future vitamin E publications. This review provides an update on vitamin E therapeutic potentials, protective effects and modes of action beyond cancer, with comparison of tocopherols against tocotrienols. With the concerted efforts in synthesizing novel vitamin E analogues and clinical pharmacology of vitamin E, it is likely that certain vitamin E isoform(s) will be therapeutic agents against human diseases besides cancer.

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