Gamma-tocotrienol induced apoptosis is associated with unfolded protein response in human breast cancer cells

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Abstract

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.

Introduction

Vitamin E includes a family of lipophilic micronutrients consisting of four tocopherols (Ts) and four tocotrienols (T3s) (α, β, γ and δ) that consist of a chromanol ring and a side chain. Both Ts and T3s are found in various components of the human diet. Tocopherols are present primarily in nuts and vegetable oils, while T3s are minor plant constituents especially abundant in rice bran, cereal grain and palm oil. Tocopherols and T3s are well recognized for their antioxidative effects. In general, antioxidants are suggested to reduce cancer by arresting free-radical-induced DNA damage. Tocopherols have been studied in great detail for their antioxidative property and physiological relevance. However, limited studies have been performed on T3s. It is well documented that T3s possess more powerful anticancer, neuroprotective and cholesterol-lowering properties that are often not exhibited by Ts [1]. The accumulation of T3s in the cells is much greater than Ts; this might be one of the reasons that T3s have more significant physiological effects than Ts [2]. Tocotrienols, particularly γ-T3, suppress the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an enzyme involved in cholesterol biosynthesis in the liver, resulting in less cholesterol being manufactured by the liver cells and an overall reduction of plasma cholesterol levels [1], [3]. γ-T3 possesses a hormone-like natriuretic function and can potentially prevent hypertension and cardiovascular disease caused by high salt intake [4].

Tocotrienols have been shown to inhibit the growth of various cancer cells without affecting the growth of normal cells [5], [6]. In breast cancer cells, γ-T3s induce apoptosis irrespective of their estrogen response status [7], [8], [9]. Although T3s are likely to be one of the powerful cancer chemotherapeutic/preventive agents in the human diet, their exact mechanisms of action on cell death and other inhibitory pathways are unknown. Gene expression profiling in estrogen receptor positive, p53 wild-type MCF-7 and estrogen receptor-negative, p53 mutant MDA-MB 231 cells treated with tocotrienol-rich fraction (TRF) of palm oil suggested different mechanisms in the two cell lines [10]. Lipid peroxidation is one of the mechanisms suggested for its antiproliferative action [11]. Other mechanisms involve modulation of various signaling pathways including apoptosis by caspase-8 activation and mitochondrial dependency [12], [13], inhibition of cell proliferation [6], down-regulation of cyclins [14], reduction in the Pl3K/PDK-1/Akt signaling and NFκb activity [15], [16] and modulation of p53, Bax/Bcl2 [17]. Estrogens are known to be involved in breast carcinogenesis. Tocotrienols have been demonstrated as antioxidants against the E2-epoxide-induced breast cancer carcinogenesis, whereas α-tocopherol was found to be less effective [18]. The effects of γ-T3 have also been studied in other cancers such as colon and prostate cancer where γ-T3 has been found to modulate multiple signaling pathways and induce apoptotic cell death. Activation of p53 has been reported in RKO human colon carcinoma cells in response to TRF [17]. Recently, Yap and colleagues reported the modulation of ID family proteins and mesenchymal markers in prostate and breast cancer cells in response to γ-T3 [19], [20].

In the present study, we examined the whole genome transcription in MCF-7 breast cancer cells when exposed to γ-T3. We demonstrate that γ-T3-induced apoptosis is associated with induction of early response genes and ER stress transcriptional response in MCF-7 cells. We have characterized the ER stress response induced by γ-T3 in MCF-7 and MDA-MB 231 breast cancer cells.

Section snippets

Cell culture and media

Human breast cancer cells (MCF-7 and MDA-MB 231) and immortalized normal human breast mammary MCF-10A cells were obtained from Lombardi Comprehensive Cancer Center cell repository and grown in Dulbecco's modified Eagle medium supplemented with 5% heat-inactivated fetal bovine serum and 25 μg/ml gentamicin (Invitrogen, Carlsbad, CA, USA).

Chemicals, reagents and antibodies

Tocotrienols (>95% pure) were from EISAI Corporation (Woodcliff Lake, NJ, USA) and Carotech (Edison, NJ, USA) and dissolved in dimethyl sulfoxide (DMSO). Other

Effects of α- and γ-T3s on the proliferation of human breast cancer cells

We compared the effects of α- and γ-T3 on the viability of MCF-7 and MDA-MB 231 cells using WST-1 assay. Both α- and γ-T3 inhibited cell proliferation in a dose-dependent manner when treated with 10–40 μM of each T3 for 24 h (Fig. 1A, B). γ-T3 exhibited a slightly better cell growth inhibitory effect at 20–40 μM as compared with α-T3 in both the cell lines (P<.05). The inhibitory effect of γ-T3 on MCF-7 and MDA-MB 231 cells was also confirmed by trypan blue dye exclusion assay (data not shown).

Discussion

Tocotrienols have gained attention due to their powerful anticancer, neuroprotective and cholesterol-lowering properties not exhibited by Ts [1]. The accumulation of T3s in the cells is much greater than Ts; this might be one of the reasons that T3s have more significant effects than Ts [2]. Tocotrienols inhibit the growth of various cancer cell lines without affecting the growth of normal cells [5], [6]; our results confirmed that, at higher concentrations of γ-T3, only the growth of breast

Acknowledgments

The studies were conducted using the Tissue Culture, Flow Cytometry and Cell Sorting and Macromolecular Analysis Shared Resources of the Lombardi Comprehensive Cancer Center. We thank Karen Creswell, Xiaojun Zou and James Li for providing excellent technical assistance.

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  • Cited by (0)

    Financial support (DK): NCI UDC-LCCC U56, UDC Agricultural Experiment Station, MBRS SCORE, NSF HBCU-UP program.

    1

    These authors contributed equally.

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