The highly malignant +SA mouse mammary epithelial cells were used as the model cell line over the years to establish the anticancer activity of tocotrienols. Tocotrienols, however, have poor oral bioavailability and were therefore entrapped into parenteral nanoemulsions for parenteral administration. The objective of this work was to test whether the activity of tocotrienols in lipid nanoemulsions against the +SA cells was retained. A secondary objective was to test whether stabilizing the nanoemulsions with poloxamer or sodium oleate would affect their activity. Nanoemulsions were found to be significantly more potent than tocotrienol/albumin conjugate. The IC50 values of the poloxamer and sodium oleate nanoemulsions were 3 and 6 microM, respectively, whereas the IC50 value of the conjugate was 10 microM. The antiproliferative activity of the nanoemulsions was also found to inversely correlate with particle size. No activity was observed with nanoemulsions loaded with alpha-tocopherol or vehicle, which confirmed the cytotoxic activity of tocotrienols and the potential use of nanoemulsions in cancer therapy.
Breast Cancer
Synergistic Antiproliferative Effects of Combined γ -Tocotrienol and PPAR γ Antagonist Treatment Are Mediated through PPAR γ -Independent Mechanisms in Breast Cancer Cells.
Malaviya A, Sylvester PW.
Previous findings showed that the anticancer effects of combined γ -tocotrienol and peroxisome proliferator activated receptor γ (PPAR γ ) antagonist treatment caused a large reduction in PPAR γ expression. However, other studies suggest that the antiproliferative effects of γ -tocotrienol and/or PPAR γ antagonists are mediated, at least in part, through PPAR γ -independent mechanism(s). Studies were conducted to characterize the role of PPAR γ in mediating the effects of combined treatment of γ -tocotrienol with PPAR γ agonists or antagonists on the growth of PPAR γ negative +SA mammary cells and PPAR γ -positive and PPAR γ -silenced MCF-7 and MDA-MB-231 breast cancer cells. Combined treatment of γ -tocotrienol with PPAR γ antagonist decreased, while combined treatment of γ -tocotrienol with PPAR γ agonist increased, growth of all cancer cells. However, treatment with high doses of 15d-PGJ2, an endogenous natural ligand for PPAR γ , had no effect on cancer cell growth. Western blot and qRT-PCR studies showed that the growth inhibitory effects of combined γ -tocotrienol and PPAR γ antagonist treatment decreased cyclooxygenase (COX-2), prostaglandin synthase (PGDS), and prostaglandin D2 (PGD2) synthesis. In conclusion, the anticancer effects of combined γ -tocotrienol and PPAR γ antagonists treatment in PPAR γ negative/silenced breast cancer cells are mediated through PPAR γ -independent mechanisms that are associated with a downregulation in COX-2, PGDS, and PGD2 synthesis.
γ-Tocotrienol-induced autophagy in malignant mammary cancer cells.
Tiwari RV, Parajuli P, Sylvester PW.
γ-Tocotrienol, a member of the vitamin E family of compounds, displays potent antiproliferative and cytotoxic effects in a variety of cancer cell types at treatment doses that have little or no effect on normal cell viability or growth. Autophagy is a tightly regulated lysosomal self-digested process that can either promote cell survival or programmed cell death, but the role of autophagy in mediating γ-tocotrienol-induced cytotoxicity in breast cancer is not presently completely understood. Mouse (+SA) and human (MCF-7 and MDA-MD-231) mammary tumor cells lines were exposed to 0-40 µmol/L γ-tocotrienol for a 24 h treatment period. γ-Tocotrienol treatment caused a relatively large increase in the accumulation of monodansylcadaverine (MDC)-labeled vacuoles, a marker of autophagosome formation, in all tumor cell lines. Results also showed that γ-tocotrienol treatment induced an increased conversion of microtubule-associated protein, 1A/1B-light chain 3, from its cytosolic form (LC3B-I) to its lipidated form (LC3B-II), increased Beclin-1 levels, and increased acridine orange staining as determined by flow cytometry analysis, providing further evidence of γ-tocotrienol-induced autophagy in these mammary cancer cell lines. In contrast, similar treatment with γ-tocotrienol was not found to increase autophagy marker expression in immortalized mouse (CL-S1) and human (MCF-10 A) normal mammary epithelial cell lines. Treatment with γ-tocotrienol also caused a reduction in PI3K/Akt/mTOR signaling and a corresponding increase in the Bax/Bcl-2 ratio, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) levels in these cancer cell lines, suggesting that γ-tocotrienol-induced autophagy may be involved in the initiation of apoptosis. In summary, these findings demonstrate that the cytotoxic effects of γ-tocotrienol are associated with the induction of autophagy in a mouse and human mammary cancer cells.
Tocotrienol-adjuvanted dendritic cells inhibit tumor growth and metastasis: a murine model of breast cancer.
Abdul Hafid SR, Chakravarthi S, Nesaretnam K, Radhakrishnan AK.
Tocotrienol-rich fraction (TRF) from palm oil is reported to possess anti-cancer and immune-enhancing effects. In this study, TRF supplementation was used as an adjuvant to enhance the anti-cancer effects of dendritic cells (DC)-based cancer vaccine in a syngeneic mouse model of breast cancer. Female BALB/c mice were inoculated with 4T1 cells in mammary pad to induce tumor. When the tumor was palpable, the mice in the experimental groups were injected subcutaneously with DC-pulsed with tumor lysate (TL) from 4T1 cells (DC+TL) once a week for three weeks and fed daily with 1 mg TRF or vehicle. Control mice received unpulsed DC and were fed with vehicle. The combined therapy of using DC+TL injections and TRF supplementation (DC+TL+TRF) inhibited (p<0.05) tumor growth and metastasis. Splenocytes from the DC+TL+TRF group cultured with mitomycin-C (MMC)-treated 4T1 cells produced higher (p<0.05) levels of IFN-γ and IL-12. The cytotoxic T-lymphocyte (CTL) assay also showed enhanced tumor-specific killing (p<0.05) by CD8(+) T-lymphocytes isolated from mice in the DC+TL+TRF group. This study shows that TRF has the potential to be used as an adjuvant to enhance effectiveness of DC-based vaccines.
Combined γ-tocotrienol and Met inhibitor treatment suppresses mammary cancer cell proliferation, epithelial-to-mesenchymal transition and migration.
Ayoub NM, Akl MR, Sylvester PW
Cell Prolif. 2013 Aug 23. doi: 10.1111/cpr.12059. [Epub ahead of print]
SUMMARY
OBJECTIVES:
Dysregulation of Met signalling is associated with malignant transformation. Combined treatment has been shown to reduce Met activation and mammary tumour cell proliferation. Experiments here, were conducted to determine mechanisms involved in mediating anti-cancer effects of combined γ-tocotrienol and SU11274 (Met inhibitor) treatment in various mammary cancer cell lines.
MATERIALS AND METHODS:
Treatment effects on mouse (+SA) and human (MCF-7, and MDA-MB-231) mammary cancer cell lines, and normal mouse (CL-S1) and human (MCF10A) mammary epithelial cell lines were compared. Cell proliferation and survival were determined by MTT assay and Ki-67 staining; protein expression was determined by western blot analysis. Immunofluorescence staining was also used to characterize expression and localization of multiple epithelial and mesenchymal markers. Cell migration was determined using a wound-healing assay.
RESULTS:
Combined treatment with γ-tocotrienol and SU11274 resulted in synergistic inhibition of +SA, MCF-7, and MDA-MB-231, but not CL-S1 or MCF10A cell growth that was associated with reduction in Akt STAT1/5 and NFκB activation and corresponding blockade in epithelial-to-mesenchymal transition, as indicated by increased expression of E-cadherin, β-catenin, and cytokeratins 8/18 (epithelial markers) and corresponding reduction in vimentin (mesenchymal marker) and reduction in cancer cell motility.
CONCLUSIONS:
Suggest that combined γ-tocotrienol and Met inhibitor treatment may provide benefit in treatment of breast cancers characterized by aberrant Met activity.
Effect of annatto-tocotrienols supplementation on the development of mammary tumors in HER-2/neu transgenic mice
Pierpaoli, E.,Viola, V.,Barucca, A.,Orlando, F.,Galli, F.,Provinciali, M.
Carcinogenesis 2013;34(6):1352-60
Tocotrienols (T3), the lesser known isomers of vitamin E, have been reported to possess anticancer activity both in in vitro and in vivo experimental models of rodents transplanted with parental tumors or treated with carcinogens. We investigated the effects of dietary supplementation with annatto-T3 (90% delta-T3 and 10% gamma-T3) on the spontaneous development of mammary tumors in HER-2/neu transgenic mice. Underlying mechanisms of the antitumor effect were evaluated by studying apoptosis, senescent-like growth arrest, immune modulation, oxidative effect and the expression of HER-2/neu in tumoral mammary glands of transgenic mice and in vitro in human and mice tumor cell lines. Annatto-T3 supplementation delayed the development of mammary tumors, reducing the number and size of mammary tumor masses and those of lung metastases. In annatto-T3-supplemented mice, both apoptosis and senescent-like growth arrest of tumor cells were increased in mammary glands while no immune modulation was observed. In vitro, a dose-dependent inhibition of cell growth, increased apoptosis and senescent-like growth arrest and a time-dependent accumulation of reactive oxygen species were observed in tumor cells treated with annatto-T3 or purified delta-T3. Annatto-T3 reduced both HER-2/neu mRNA and p185(HER-2/neu) protein in tumors and in tumor cell lines. The results show that the antitumor effect of annatto-T3 supplementation in HER-2/neu transgenic mice is mainly related to the direct induction of oxidative stress, senescent-like growth arrest and apoptosis of tumor cells rather than to an immune modulation.
Tocotrienols promote apoptosis in human breast cancer cells by inducing poly(ADP-ribose) polymerase cleavage and inhibiting nuclear factor kappa-B activity
Loganathan, R.,Selvaduray, K. R.,Nesaretnam, K.,Radhakrishnan, A. K.
Cell Prolif 2013;46(2):203-13
OBJECTIVES: Tocotrienols and tocopherols are members of the vitamin E family, with similar structures; however, only tocotrienols have been reported to achieve potent anti-cancer effects. The study described here has evaluated anti-cancer activity of vitamin E to elucidate mechanisms of cell death, using human breast cancer cells. MATERIALS AND METHODS: Anti-cancer activity of a tocotrienol-rich fraction (TRF) and a tocotrienol-enriched fraction (TEF) isolated from palm oil, as well as pure vitamin E analogues (alpha-tocopherol, alpha-, delta- and gamma-tocotrienols) were studied using highly aggressive triple negative MDA-MB-231 cells and oestrogen-dependent MCF-7 cells, both of human breast cancer cell lines. Cell population growth was evaluated using a Coulter particle counter. Cell death mechanism, poly(ADP-ribose) polymerase cleavage and levels of NF-kappaB were determined using commercial ELISA kits. RESULTS: Tocotrienols exerted potent anti-proliferative effects on both types of cell by inducing apoptosis, the underlying mechanism of cell death being ascertained using respective IC50 concentrations of all test compounds. There was marked induction of apoptosis in both cell lines by tocotrienols compared to treatment with Paclitaxel, which was used as positive control. This activity was found to be associated with cleavage of poly(ADP-ribose) polymerase (a DNA repair protein), demonstrating involvement of the apoptotic cell death signalling pathway. Tocotrienols also inhibited expression of nuclear factor kappa-B (NF-kappaB), which in turn can increase sensitivity of cancer cells to apoptosis. CONCLUSION: Tocotrienols induced anti-proliferative and apoptotic effects in association with DNA fragmentation, poly(ADP-ribose) polymerase cleavage and NF-kappaB inhibition in the two human breast cancer cell lines.
Eliminating drug resistant breast cancer stem-like cells with combination of simvastatin and gamma-tocotrienol
Gopalan, A., Yu, W., Sanders, B. G., Kline, K.
Cancer Lett 2013;328:285-96
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 (gammaT3) 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+gammaT3 at low doses enhanced these actions via inhibition of the mevalonate pathway. Data demonstrate that SVA and gammaT3 alone or in combination possess the ability to eliminate CSCs in drug resistant human breast cancer cells.
Mitochondrial-dependent anticancer activity of delta-tocotrienol and its synthetic derivatives in HER-2/neu overexpressing breast adenocarcinoma cells
Viola V, Ciffolilli S, Legnaioli, S et al
Biofactors; epub Jan30,2013
Anticancer activity and mitochondrial mechanism of the vitamin E form delta-tocotrienol (delta-T3) was investigated in HER-2/neu-overexpressing human SKBR3 and murine TUBO breast cancer cells. delta-T3 was confirmed to possess high cytotoxic and apoptotic activity in SKBR3 cells as compared with all natural forms of vitamin E and several synthetic forms that included novel derivatives with the same backbone of delta-T3 such as delta-tocotrienyl-succinyl amide (delta-T3AS) and the redox-active analogue delta-tocotrienyl amine (delta-T3NH2). As observed in the case of alpha-TOS, a prototypical anticancer drug derived from alpha-tocopherol, succinylation of delta-T3 enhanced citotoxicity and apoptotic activity of the vitamer. delta-T3 induced apoptosis of SKBR3 cells was associated with mitochondrial destabilization, energy failure, and unbalanced activity of stress/survival MAPKs, namely p38 and ERK1/2 pathways. An increased generation of ROS followed to such a series of early events. Enhanced activity of delta-T3 in this human carcinoma cell line was characterized by the sustained uptake and oxidative transformation to the quinone derivative delta-T3Q, thereby suggesting redox effects in SKBR3 mitochondria by this vitamer. Viability and uptake data show a different pattern of responses in TUBO cells with higher response to synthetic derivatives of delta-T3 than in SKBR3 cells. In conclusion, synthetic derivatives of delta-T3 with enhanced apoptotic activity in breast carcinoma cells are investigated for the first time in this study also describing mechanistic aspects of mitochondrial effects of delta-T3. Further investigation in preclinical models of HER2/neu-high breast adenocarcinoma is underway to identify other and more effective forms of VE in this type of cancer.
Sesamin synergistically potentiates the anticancer effects of γ-tocotrienol in mammary cancer cell lines
Akl MR, Ayoub NM, Abuasal BS, Kaddoumi A, Sylvester PW.
Fitoterapia. 2013 Jan;84:347-59.
Summary
γ-Tocotrienol and sesamin are phytochemicals that display potent anticancer activity. Since sesamin inhibits the metabolic degradation of tocotrienols, studies were conducted to determine if combined treatment with sesamin potentiates the antiproliferative effects of γ-tocotrienol on neoplastic mouse (+ SA) and human (MCF-7 and MDA-MB-231) mammary cancer cells. Results showed that treatment with γ-tocotrienol or sesamin alone induced a significant dose-responsive growth inhibition, whereas combination treatment with these agents synergistically inhibited the growth of + SA, MCF-7 and MDA-MB-231 mammary cancer cells, while similar treatment doses were found to have little or no effect on normal (mouse CL-S1 and human MCF-10A) mammary epithelial cell growth or viability. However, sesamin synergistic enhancement of γ-tocotrienol-induced anticancer effects was not found to be mediated from a reduction in γ-tocotrienol metabolism. Rather, combined treatment with subeffective doses of γ-tocotrienol and sesamin was found to induce G1 cell cycle arrest, and a corresponding decrease in cyclin D1, CDK2, CDK4, CDK6, phospho-Rb, and E2F1 levels, and increase in p27 and p16 levels. Additional studies showed that the antiproliferative effect of combination treatment did not initiate apoptosis or result in a decrease in mammary cancer cell viability. Taken together, these findings indicate that the synergistic antiproliferative action of combined γ-tocotrienol and sesamin treatment in mouse and human mammary cancer cells is cytostatic, not cytotoxic, and results from G1 cell cycle arrest.