**2. Vitamin E and breast cancer**

Epidemiological studies have shown that diet and nutrition can play a major role in cancer development and progression. It has been suggested that approximately 30–35% of cancer morbidity and mortality might be prevented with suitable adjustment of nutrition, and up to one third of all the cancers in the United States can be avoided by increasing the consumption of fruits and vegetables in the daily diet [20]. Vitamin E is a generic term that includes a family of eight naturally occurring compounds that are further divided into two subgroups known as tocotrienols and tocopherols. Tocotrienols are relatively rare and found only a few natural sources, such as palm oil, rice bran oil, and annatto bean, while tocopherols are much more abundant and found in a wide variety of foods, such as nuts, whole grains, dark green vegetables, egg yolk, and various vegetable oils [21–24]. The relative levels of tocopherol and tocotrienol in various dietary oil and fats are shown in **Table 1**.

The chemical structure of all vitamin E isoforms are very similar and characterized by a long phytyl chain linked to a chroman ring structure methylated to varying degrees at the 5, 7, and 8 positions. The four isoforms in each subclass are classified as α-, β-, γ-, and δ-tocotrienol γ-Tocotrienol Reversal of Epithelial-to-Mesenchymal Transition in Human Breast Cancer Cells… http://dx.doi.org/10.5772/intechopen.78273 85


**Table 1.** Vitamin E levels (mg/L) in common dietary oils [21].

**1. Introduction**

84 Vitamin E in Health and Disease

and Hedgehog signaling pathways.

**2. Vitamin E and breast cancer**

tocotrienol in various dietary oil and fats are shown in **Table 1**.

Breast cancer is the second leading cause of death in women, and it originates from malignant breast cancer cells displaying unregulated growth to produce a tumor mass [1, 2]. Several cellular mechanisms are dysregulated in breast tumor cells, including the canonical Wnt and Hedgehog signaling pathways, which play an important role in promoting oncogenic proliferation, survival, motility, invasion, and epithelial-to-mesenchymal transition (EMT) [3]. Although these events are complex and poorly understood, recent findings show that specialized cell membrane microdomains known as lipid rafts are involved in mediating membrane receptor activation and signal transduction. Lipid rafts are solid platforms in the plasma membrane that consist of cholesterol and sphingolipids. Lipid rafts are essential for cellular signaling by recruiting transmembrane receptors with adaptor and signaling proteins from non-rafts to the raft area of the cell membrane [4–6]. In the case of canonical Wnt and Hedgehog signaling, low-density lipoprotein receptor-related protein 6 (LRP6) and patched (PTCH2), the main receptors for activation of these signaling pathways, were shown to be primarily located in the lipid raft microdomain [7–9]. Lipid rafts have been shown to be essential for Hedgehog signal transduction [10]. γ-Tocotrienol is a natural vitamin E isoform that displays potent anticancer activities [11–13]. Previous reports have clearly shown that γ-tocotrienol exerts antiproliferative and apoptotic activity against neoplastic mammary epithelial cells at treatment doses that had little or no effect on normal cell growth and viability [14, 15]. The anticancer effects of γ-tocotrienol appear to be mediated through a variety of intracellular signaling mechanism [16–18]. Recently, γ-tocotrienol was found to disrupt lipid raft integrity and attenuation of receptor signaling transduction [19]. This chapter will focus of experimental evidence demonstrating γ-tocotrienol reversal of EMT is mediated through the inhibition of the canonical Wnt

Epidemiological studies have shown that diet and nutrition can play a major role in cancer development and progression. It has been suggested that approximately 30–35% of cancer morbidity and mortality might be prevented with suitable adjustment of nutrition, and up to one third of all the cancers in the United States can be avoided by increasing the consumption of fruits and vegetables in the daily diet [20]. Vitamin E is a generic term that includes a family of eight naturally occurring compounds that are further divided into two subgroups known as tocotrienols and tocopherols. Tocotrienols are relatively rare and found only a few natural sources, such as palm oil, rice bran oil, and annatto bean, while tocopherols are much more abundant and found in a wide variety of foods, such as nuts, whole grains, dark green vegetables, egg yolk, and various vegetable oils [21–24]. The relative levels of tocopherol and

The chemical structure of all vitamin E isoforms are very similar and characterized by a long phytyl chain linked to a chroman ring structure methylated to varying degrees at the 5, 7, and 8 positions. The four isoforms in each subclass are classified as α-, β-, γ-, and δ-tocotrienol or tocopherol. Tocotrienols differ from tocopherols only in that they contain an unsaturated, whereas tocopherols contain a saturated phytyl tail. **Figure 1** shows the chemical structures of different isoforms of tocotrienols.

Interestingly, numerous studies have shown that tocotrienols, but not tocopherols, have selective antiproliferative and apoptotic effects against various forms of breast cancer, while have little effect on normal mammary epithelial cell growth or function [14, 15, 25]. The anticancer

**Figure 1.** General chemical structure of the different tocotrienols isoforms.

potency of different tocotrienol isoforms was determined to be characterized as δ-tocotrienol ≥ γ-tocotrienol > α-tocotrienol > β-tocotrienol [11, 15, 26]. The anticancer effects of tocotrienols were discovered in nutritional studies that investigated the role of high-dietary fat consumption on the development of mammary tumorigenesis in laboratory animals. These studies showed that diets containing high levels of palm oil inhibited the carcinogen-induced mammary cancer in rats [27]. Additional studies showed that palm oil diets stripped of tocotrienol no longer displays their protective effect against mammary tumorigenesis.

effects γ-tocotrienol has on numerous signaling pathways [19]. Molecular targets associated

γ-Tocotrienol Reversal of Epithelial-to-Mesenchymal Transition in Human Breast Cancer Cells…

http://dx.doi.org/10.5772/intechopen.78273

87

**Figure 2** shows the effects of γ-tocotrienol treatment on the growth of malignant and normal human breast cancer cells. Results show that exposure various doses of γ-tocotrienol induced a dose-dependent inhibition in the growth of the highly malignant MDA-MB-231 breast cancer cells, as compared to cells in the vehicle-treated control group in **Figure 2A**. The IC50 dose γ-tocotrienol in these studies was found to be approximately 5 μM. However, treatment with similar or even higher doses of γ-tocotrienol on immortalized normal MCF-10A mammary epithelial cell line was found to have little or no effect on cell growth or viability (**Figure 2B**) [14].

**Table 2.** Summary of some of the molecular targets associated with mediating the anticancer effects of tocotrienols.

**Figure 2.** γ-Tocotrienol effects on the growth of the highly malignant MDA-MB-231 human breast cancer cells and the immortalized normal MCF-10A human mammary epithelial cells. MDA-MB-231 and MCF-10A cells were initially

media containing 0–30 μM doses of γ-tocotrienol over a 4-day culture period. The viable cell number was determined by using the MTT colorimetric assay. Vertical bars show mean cell number ± SEM in each treatment group. (\**P* < 0.05) as

cells/well (6 wells/group) in 96-well culture plates and maintained on serum-free defined

seeded at a density of 1 × 10<sup>4</sup>

compared with cells in their respective vehicle-treated control groups.

with tocotrienol anticancer activity are shown in **Table 2**.

**Molecular target References** PI3K/Akt [29] PKCα [32] MAPK [33] Cell cycle [13] Mevalonate pathway [34, 35] Glycolysis [12] Angiogenesis [36] EMT [37] Lipid rafts [19]

During the past decade, tocotrienols have received a great deal of attention because of their potential value in the prevention and treatment of breast cancer. Tocotrienols have been shown to inhibit multiple intracellular signaling pathways in cancer cells [15, 28]. Specifically, tocotrienols have been found to suppress EGF-dependent mitogenic signaling in neoplastic and normal mammary epithelial cells by significantly inhibiting activity of the phosphatidylinositol-4, 5-bisphosphate-3-kinase/protein kinase B (PI3K/Akt) pathway [29]. Other studies have shown that γ-tocotrienol treatment induced a dose and time-dependent inhibition of EGF-dependent Akt phosphorylation (activation) in mammary tumor cells, and these effects were not found to be associated with an increase in tensin homolog (PTEN) or protein phosphatase 2 A (PP2A) activity [30]. γ-Tocotrienol was also found to decrease activity of signaling proteins downstream of Akt, such as inhibiting the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) by suppressing the activation of inhibitor of nuclear factor kappa kinase alpha and beta (IKKα and IKKβ), enzymes associated with induction of the NFκB activation [30]. Inhibition of NFκB transcription is associated with a suppression in cell proliferation and survival [31]. Additional studies have shown that the antiproliferative effects of tocotrienols is associated with an inhibition of protein kinase C alpha (PKCα) activation in breast cancer cells [32]. In addition, mitogen activated protein kinase (MAPK) has also been shown to be a target of γ-tocotrienol anticancer activity. Studies have indicated that γ-tocotrienol induced inhibition of EGF-dependent proliferation of preneoplastic CL-S1 mouse mammary epithelial cells resulted from an inhibition of G-protein-mediated activation of adenylyl cyclase, cyclic adenosine monophosphate (cAMP) production, as well as a reduction in phosphorylated (activated) extracellular signal-regulated kinase 1/2 (ERK1 and ERK2) [33]. In addition to the inhibition of mitogenic signaling, γ-tocotrienol is known to inhibit numerous vital cellular functions including inhibition of cell cycle progression [13], mevalonate pathway [34, 35], glycolysis [12], angiogenesis [36], and epithelial mesenchymal transition (EMT) [37].

Lipid rafts are distinct structures within the cell membrane that are enriched with sphingolipids, cholesterol, and acyl fatty acid chains that act to form a very rigid microdomain. Lipid rafts exist in two different forms: "planar lipid rafts," which are referred to as "non-caveolar" and caveolae lipid rafts. Planar rafts are characterized as non-invaginated microdomains lacking specific morphological features. In contrast, caveolae lipid rafts are tube-like invaginations of the plasma membrane characterized by specific scaffolding proteins or caveolins [4]. Some proteins are essential to membrane raft development and their role can be seen as constitutive components of rafts. One of the important proteins serving scaffolding functions in the caveolar raft is caveolin 1 (Cav1), a classical hairpin protein that plays a role in caveolae-mediated signaling, endocytosis, and transport [4]. Recent studies have shown that tocotrienols act to disrupt lipid raft integrity and disrupt plasma receptor membrane receptor activation and signal transduction. These findings provide evidence to explanation the wide range of inhibitory effects γ-tocotrienol has on numerous signaling pathways [19]. Molecular targets associated with tocotrienol anticancer activity are shown in **Table 2**.

potency of different tocotrienol isoforms was determined to be characterized as δ-tocotrienol ≥ γ-tocotrienol > α-tocotrienol > β-tocotrienol [11, 15, 26]. The anticancer effects of tocotrienols were discovered in nutritional studies that investigated the role of high-dietary fat consumption on the development of mammary tumorigenesis in laboratory animals. These studies showed that diets containing high levels of palm oil inhibited the carcinogen-induced mammary cancer in rats [27]. Additional studies showed that palm oil diets stripped of tocotrienol

During the past decade, tocotrienols have received a great deal of attention because of their potential value in the prevention and treatment of breast cancer. Tocotrienols have been shown to inhibit multiple intracellular signaling pathways in cancer cells [15, 28]. Specifically, tocotrienols have been found to suppress EGF-dependent mitogenic signaling in neoplastic and normal mammary epithelial cells by significantly inhibiting activity of the phosphatidylinositol-4, 5-bisphosphate-3-kinase/protein kinase B (PI3K/Akt) pathway [29]. Other studies have shown that γ-tocotrienol treatment induced a dose and time-dependent inhibition of EGF-dependent Akt phosphorylation (activation) in mammary tumor cells, and these effects were not found to be associated with an increase in tensin homolog (PTEN) or protein phosphatase 2 A (PP2A) activity [30]. γ-Tocotrienol was also found to decrease activity of signaling proteins downstream of Akt, such as inhibiting the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) by suppressing the activation of inhibitor of nuclear factor kappa kinase alpha and beta (IKKα and IKKβ), enzymes associated with induction of the NFκB activation [30]. Inhibition of NFκB transcription is associated with a suppression in cell proliferation and survival [31]. Additional studies have shown that the antiproliferative effects of tocotrienols is associated with an inhibition of protein kinase C alpha (PKCα) activation in breast cancer cells [32]. In addition, mitogen activated protein kinase (MAPK) has also been shown to be a target of γ-tocotrienol anticancer activity. Studies have indicated that γ-tocotrienol induced inhibition of EGF-dependent proliferation of preneoplastic CL-S1 mouse mammary epithelial cells resulted from an inhibition of G-protein-mediated activation of adenylyl cyclase, cyclic adenosine monophosphate (cAMP) production, as well as a reduction in phosphorylated (activated) extracellular signal-regulated kinase 1/2 (ERK1 and ERK2) [33]. In addition to the inhibition of mitogenic signaling, γ-tocotrienol is known to inhibit numerous vital cellular functions including inhibition of cell cycle progression [13], mevalonate pathway [34, 35],

no longer displays their protective effect against mammary tumorigenesis.

86 Vitamin E in Health and Disease

glycolysis [12], angiogenesis [36], and epithelial mesenchymal transition (EMT) [37].

Lipid rafts are distinct structures within the cell membrane that are enriched with sphingolipids, cholesterol, and acyl fatty acid chains that act to form a very rigid microdomain. Lipid rafts exist in two different forms: "planar lipid rafts," which are referred to as "non-caveolar" and caveolae lipid rafts. Planar rafts are characterized as non-invaginated microdomains lacking specific morphological features. In contrast, caveolae lipid rafts are tube-like invaginations of the plasma membrane characterized by specific scaffolding proteins or caveolins [4]. Some proteins are essential to membrane raft development and their role can be seen as constitutive components of rafts. One of the important proteins serving scaffolding functions in the caveolar raft is caveolin 1 (Cav1), a classical hairpin protein that plays a role in caveolae-mediated signaling, endocytosis, and transport [4]. Recent studies have shown that tocotrienols act to disrupt lipid raft integrity and disrupt plasma receptor membrane receptor activation and signal transduction. These findings provide evidence to explanation the wide range of inhibitory **Figure 2** shows the effects of γ-tocotrienol treatment on the growth of malignant and normal human breast cancer cells. Results show that exposure various doses of γ-tocotrienol induced a dose-dependent inhibition in the growth of the highly malignant MDA-MB-231 breast cancer cells, as compared to cells in the vehicle-treated control group in **Figure 2A**. The IC50 dose γ-tocotrienol in these studies was found to be approximately 5 μM. However, treatment with similar or even higher doses of γ-tocotrienol on immortalized normal MCF-10A mammary epithelial cell line was found to have little or no effect on cell growth or viability (**Figure 2B**) [14].


**Table 2.** Summary of some of the molecular targets associated with mediating the anticancer effects of tocotrienols.

**Figure 2.** γ-Tocotrienol effects on the growth of the highly malignant MDA-MB-231 human breast cancer cells and the immortalized normal MCF-10A human mammary epithelial cells. MDA-MB-231 and MCF-10A cells were initially seeded at a density of 1 × 10<sup>4</sup> cells/well (6 wells/group) in 96-well culture plates and maintained on serum-free defined media containing 0–30 μM doses of γ-tocotrienol over a 4-day culture period. The viable cell number was determined by using the MTT colorimetric assay. Vertical bars show mean cell number ± SEM in each treatment group. (\**P* < 0.05) as compared with cells in their respective vehicle-treated control groups.
