Present study shows that drug resistant human breast cancer cells are enriched in cancer stem-like cells (CSCs) and express elevated levels of Stat-3 signaling mediators, which contribute to CSC enrichment. Simvastatin (SVA) and gamma-tocotrienol (γT3) eliminate enriched CSCs and suppress expression of Stat-3 signaling mediators via inhibition of the mevalonate pathway and activation of de novo ceramide synthesis pathway, respectively. Combination of SVA+γT3 at low doses enhanced these actions via inhibition of the mevalonate pathway. Data demonstrate that SVA and γT3 alone or in combination possess the ability to eliminate CSCs in drug resistant human breast cancer cells.

Scope: This study further examines mechanisms involved in the pro-apoptotic action of gamma-tocopherol (γT) and gamma-tocotrienol (γT3) in human breast cancer cell lines.

Methods And Results: γT upregulates phospho-JNK (pJNK), CCAAT/enhancer-binding protein homologous protein (CHOP), and death receptor-5 (DR5) protein expression as detected by Western blot assays. siRNA knockdown of JNK, CHOP, or DR5 shows that γT-induced apoptosis is JNK/CHOP/DR5 signaling dependent, which is similar to γT3-mediated apoptotic signaling. Furthermore, both γT and γT3 induce increased levels of cellular ceramides and dihydroceramides as determined by LC-MS/MS analyses. Inhibition of de novo ceramide synthesis using chemical inhibitors blocked the ability of γT and γT3 to induce apoptosis as detected by Annexin V-FITC/PI assay and to activate JNK/CHOP/DR5 pro-apoptotic signaling thereby demonstrating the involvement of de novo ceramide synthesis in γT- and γT3-induced apoptosis.

Conclusion: Taken together, data show that both γT and γT3 induce apoptosis via de novo ceramide synthesis dependent activation of JNK/CHOP/DR5 pro-apoptotic signaling.

Scope: Docosahexaenoic acid (DHA) has been shown to exhibit anticancer actions in vitro and in vivo in a variety of cancers. Here, we investigated the role for DHA in inducing apoptosis in triple-negative breast cancer (TNBC) and studied the mechanisms of action.

Methods and Results: DHA induces apoptosis as detected byAnnexinV-FITC/PI assay as well as induces cleavage of caspase-8 and -9, endoplasmic reticulum stress (ERS), and elevated levels of death receptor-5 (DR5) protein expression as detected by western blot assays. Chemical inhibitors of caspase-8 and -9 and small interfering RNAs (siRNAs) show DHA to induce ERS/CHOP/DR5-mediated caspase-8 and -9 dependent apoptosis. Furthermore, DHA induces elevated cellular levels of reactive oxygen species (ROS) and antioxidant; RRR-α-tocopherol (_T) blocked DHA-induced apoptotic events. In contrast to the antagonistic impact of   αT, gamma tocotrienol(γT3) was demonstrated to cooperate with DHA in inducing apoptotic events in TNBC cells.

Conclusion: Data, for the first time, demonstrate thatDHAinduces apoptosis in TNBC cells via activation of ERS/CHOP/DR5-mediated caspase-8 and -9 dependent pro-apoptotic events, and that different forms of vitamin E exhibit distinct effects on DHA-induced apoptosis; namely, inhibition by αT and enhancement by γT3.

Gamma-tocotrienol (γ-T3) is a member of the vitamin E family. Tocotrienols (T3s) are powerful antioxidants and possess anticancer, neuroprotective and cholesterol-lowering properties. Tocotrienols inhibit the growth of various cancer cell lines without affecting normal cells. Less is known about the exact mechanisms of action of T3s on cell death and other growth inhibitory pathways. In the present study, we demonstrate that γ-T3 induces apoptosis in MDA-MB 231 and MCF-7 breast cancer cells as evident by PARP cleavage and caspase-7 activation. Gene expression analysis of MCF-7 cells treated with γ-T3 revealed alterations in the expression of multiple genes involved in cell growth and proliferation, cell death, cell cycle, cellular development, cellular movement and gene expression. Further analysis of differentially modulated genes using Ingenuity Pathway Analysis software suggested modulation of canonical signal transduction or metabolic pathways such as NRF-2-mediated oxidative stress response, TGF-β signaling and endoplasmic reticulum (ER) stress response. Analysis of ER-stress-related proteins in MCF-7 and MDA-MB 231 cells treated with γ-T3 demonstrated activation of PERK and pIRE1α pathway to induce ER stress. Activating transcription factor 3 (ATF3) was identified as the most up-regulated gene (16.8-fold) in response to γ-T3. Activating transcription factor 3 knockdown using siRNA suggested an essential role of ATF3 in γ-T3-induced apoptosis. In summary, we demonstrate that γ-T3 modulates ER stress signaling and have identified ATF3 as a molecular target for γ-T3 in breast cancer cells.

Anti-angiogenic therapy is widely being used to halt tumour angiogenesis. In this study, the anti-angiogenic activity of palm tocotrienol-rich fraction (TRF) and its individual components (γ- and δ-tocotrienol) were first investigated in vitro in human umbilical vein endothelial cells (HUVEC) and 4T1 mouse mammary cancer cells. Results showed reduced levels of Interkeukin (IL)-8 and IL-6, two pro-angiogenic cytokines in HUVEC treated with palm tocotrienols compared with α-tocopherol (α-T) and control cells (P < 0.05). The production of IL-8 and IL-6 was lowest in δ-tocotrienol (δ-T3)-treated cells followed by γ-tocotrienol (γ-T3) and TRF. There was significant (P < 0.05) reduction in IL-8 and vascular endothelial growth factor (VEGF) production in 4T1 cells treated with TRF or δ-T3. There was decreased expression of VEGF and its receptors; VEGF-R1 (fms-like tyrosine kinase, Flt-1) and VEGF-R2 (Kinase-insert-domain-containing receptor, KDR/Flk-2) in tumour tissues excised from mice supplemented with TRF were observed. There was also decreased expression of VEGF-R2 in lung tissues of mice supplemented with TRF. These observations correlate with the smaller tumour size recorded in the tocotrienol-treated mice. This study confirms previous observations that palm tocotrienols exhibit anti-angiogenic properties that may inhibit tumour progression.

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Breast cancer is the second most frequent cancer affecting women worldwide after lung cancer. The toxicity factor associated with synthetic drugs has turned the attention toward natural compounds as the primary focus of interest as anticancer agents. Vitamin E derivatives consisting of the well-established tocopherols and their analogs namely tocotrienols have been extensively studied due to their remarkable biological properties. While tocopherols have failed to offer protection, tocotrienols, in particular, a-, d-, and c-tocotrienols alone and in combination have demonstrated anticancer properties. The discovery of the antiangiogenic, antiproliferative, and apoptotic effects of tocotrienols, as well as their role as an inducer of immunological functions, not only reveals a new horizon as a potent antitumor agent but also reinforces the notion that tocotrienols are indeed more than antioxidants. On the basis of a transcriptomic platform, we have recently demonstrated a novel mechanism for tocotrienol activity that involves estrogen receptor (ER) signaling. In silico simulations and in vitro binding analyses indicate a high affinity of specific forms of tocotrienols for ERb, but not for ERa. Moreover, we have demonstrated that specific tocotrienols increase ERb translocation into the nucleus which, in turn, activates the expression of estrogenresponsive genes (MIC-1, EGR-1 and Cathepsin D) in breast cancer cells only expressing ERb cells (MDA-MB-231) and in cells expressing both ER isoforms (MCF-7). The binding of specific tocotrienol forms to ERb is associated with the alteration of cell morphology, caspase-3 activation, DNA fragmentation, and apoptosis. Furthermore, a recently concluded clinical trial seems to suggest that tocotrienols in combination with tamoxifen may have the potential to extend breast cancer-specific survival.

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Objectives: Aberrant Met signalling is associated with aggressive cancer cell phenotypes. γ-tocotrienol displays potent anti-cancer activity that is associated with suppression of HER⁄ErbB receptor signalling. Experiments were conducted to investigate the effects of γ-tocotrienol treatment on HGF-dependent +SA mammary tumour cell proliferation, upon Met activation.

Materials and Methods: The +SA cells were maintained in serum-free defined media containing 10 ng ⁄ml HGF as the mitogen. Cell viability was determined using the MTT assay, western blot analysis was used to measure protein expression, and Met expression and activation were determined using immunofluorescent staining.

Results and Conclusions: Treatment with γ-tocotrienol or Met inhibitor, SU11274, significantly inhibited HGF-dependent +SA cell replication in a dose– responsive manner. Treatment with 4 lM c-tocotrienol reduced both total Met levels and HGF-induced Met autophosphorylation. In contrast, similar treatment with 5.5 lM SU11274 inhibited HGF-induced Met autophosphorylation, but had no effect on total Met levels. Combined treatment with subeffective doses of c-tocotrienol (2 lM) and SU11274 (3 lM) resulted in significant inhibition of +SA cell expansion compared to treatment with individual agents alone. These findings show, for the first time, the inhibitory effects of c-tocotrienol on Met expression and activation, and strongly suggest that c-tocotrienol treatment may provide significant health benefits in prevention and ⁄ or treatment of breast cancer, in women with deregulated HGF⁄Met signalling.

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.