**3. Epithelial-to-mesenchymal transition (EMT)**

EMT plays a major role in organogenesis, angiogenesis and cancer metastasis [38, 39]. EMT was first observed and defined in the late 1980s at Harvard University by Elizabeth Hey [40]. EMT was defined as a differentiation program by which epithelial cells lose their attachment with other epithelial cells to become more mesenchymal-like, and are able to become mobile and invade their surrounding extracellular matrix [41, 42]. Because this process is reversible [43], epithelial cells displaying a mesenchymal phenotype, are also able to re-differentiate back into their epithelial phenotype [44]. Epithelial cells displaying their normal epithelial phenotype are well-structured in single layers of cuboidal or columnar cells. They are closely attached to surrounding cells by intercellular adhesion complexes. These cells also display an apicobasal polarity with a characteristic basal basement membrane that separates the epithelium from other tissues. In contrast, epithelial cells with a mesenchymal phenotype are characterized by the absence of polarity and intercellular adhesion junctions, hallmarks that have come to define EMT [45]. During the EMT process, cells lose the attachment of β-catenin and E-cadherin, which act to tightly link and attach surrounding epithelial cells together. This loss of attachment leads to a disruption of the adherens junctions [46]. These events then allow the mesenchymal phenotype to move freely and invade the surrounding extracellular matrix. In normal conditions, EMT provides a necessary function during embryogenesis, growth and wound healing. However, aberrant EMT can result in pathological conditions such as organ fibrosis and cancer metastasis. EMT mediated metastasis of malignant breast cancer epithelial cells can often form secondary tumors in the bone or lung [46]. EMT that occurs under normal conditions, such as embryogenesis, is referred to as type 1 EMT or classical EMT [47]. However, EMT that develops during inflammation, wound healing, tissue regeneration, and organ fibrosis is referred to as type 2 EMT, whereas EMT associated with cancer metastasis is termed type 3 EMT and plays an important role in the development, growth and progression of breast cancer [47].

prominent targets are the ZEB1 and ZEB2, known as specific repressors of E-cadherin. Likewise, members of the ABC family of transporters, such as ABCB5, plays a major role in the activation of EMT [49]. Studies have shown that several signaling pathways, including the canonical Wnt pathway, the canonical Hedgehog pathway, Notch pathway, Janus kinase (JAK)/STAT pathway, and TGFβ pathway are involved in the activation of EMT [50]. Activation of these EMTinducing signaling pathways leads to the disruption of adherens junctions (desmosomes), tight junctions, and gap junctions through suppression of several proteins, such as partitioning defective 6 homolog alpha or ZEB1, which represses plakophilin, an important junctional adhesion protein [43]. These pathways can act separately or together through cross-talk to increase cancer cell migration, invasion, drug resistance, stemness, and self-renewal potential [51, 52]. Taken together, it is clearly evident that EMT is an extremely complex process and a

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**Figure 3** shows the effects of γ-tocotrienol on the expression of EMT cellular biomarkers in the highly malignant MDA-MB-231 human breast cancer cells. Western blot analysis shows that MDA-MB-231 cells in the vehicle-treated control group displayed relatively low levels of expression for the epithelial cell markers cytokeratin 8, cytokeratin 18 and E-cadherin, and corresponding high levels of expression for the mesenchymal cell markers vimentin, fibronectin and total β-catenin (**Figure 3A**). Treatment with 3–7 μM γ-tocotrienol (MDA-MB-231) induced a dose-responsive reversal in epithelial versus mesenchymal cell marker expression (**Figure 3A**). Immunocytochemistry was then performed to confirm the finding in **Figure 3A**.

**Figure 3.** γ-Tocotrienol effects on epithelial versus mesenchymal cell markers expression. (A) Whole cell lysates were prepared from cells in each treatment group for subsequent separation by polyacrylamide gel electrophoresis (35 μg/lane) followed by western blot analysis. (B) Immunocytochemical analysis was done to confirm the finding shown in A. Cells in the various treatment groups were fixed, blocked, and incubated with specific primary antibodies for cytokeratin 8, cytokeratin 18, vimentin, and total level of β-catenin followed by incubation with Alexa Fluor 488-conjugated secondary antibody. Green staining in the photomicrographs (magnification 200×) indicates positive fluorescence staining for

target proteins and the blue color represents counter staining of the cell nuclei with DAPI.

great deal more information is required to fully understand this phenomenon.

Transcription factors also play a role in the initiation of EMT. Receptor activation by various growth factors, such as hepatocyte growth factor (HGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) are involved in the activation of various transcription factors involved in EMT. Growth factor-induced activation of transcription factors include zinc finger protein snail 1 (SNAIL1), (SNAIL2), zinc finger e-box-binding homeobox 1 (ZEB1), (ZEB2), twist, forkhead box protein 1 (FOXC1), (FOXC2), transcription factor 3 (TCF3), also known as (E47), and homeobox protein goosecoid (GSC) [37, 43, 45]. EMT also plays a role in the restructuring of extracellular matrix proteins by up-regulating fibronectin, collagen, proteases like MMPs, and other remodeling enzymes. In addition, autocrine and paracrine secretion of growth factors, cytokines, and extracellular proteins can modulate cancer cells phenotype and promote EMT [37, 43, 45].

Epigenetic modification, such as acetylation or methylation of the DNA, also can play a role in the EMT activation. For example, methylation of arginine (R531) by protein arginine methyltransferases 7 (PRMT7) plays a crucial role in inducing the EMT and the promotion of migratory and invasive behavior of breast cancer cells [48]. EMT can also be activated by expression of certain miRNAs, such as micro-RNA200/205 family (miRNA200) and (miRNA205), whose prominent targets are the ZEB1 and ZEB2, known as specific repressors of E-cadherin. Likewise, members of the ABC family of transporters, such as ABCB5, plays a major role in the activation of EMT [49]. Studies have shown that several signaling pathways, including the canonical Wnt pathway, the canonical Hedgehog pathway, Notch pathway, Janus kinase (JAK)/STAT pathway, and TGFβ pathway are involved in the activation of EMT [50]. Activation of these EMTinducing signaling pathways leads to the disruption of adherens junctions (desmosomes), tight junctions, and gap junctions through suppression of several proteins, such as partitioning defective 6 homolog alpha or ZEB1, which represses plakophilin, an important junctional adhesion protein [43]. These pathways can act separately or together through cross-talk to increase cancer cell migration, invasion, drug resistance, stemness, and self-renewal potential [51, 52]. Taken together, it is clearly evident that EMT is an extremely complex process and a great deal more information is required to fully understand this phenomenon.

**3. Epithelial-to-mesenchymal transition (EMT)**

88 Vitamin E in Health and Disease

EMT plays a major role in organogenesis, angiogenesis and cancer metastasis [38, 39]. EMT was first observed and defined in the late 1980s at Harvard University by Elizabeth Hey [40]. EMT was defined as a differentiation program by which epithelial cells lose their attachment with other epithelial cells to become more mesenchymal-like, and are able to become mobile and invade their surrounding extracellular matrix [41, 42]. Because this process is reversible [43], epithelial cells displaying a mesenchymal phenotype, are also able to re-differentiate back into their epithelial phenotype [44]. Epithelial cells displaying their normal epithelial phenotype are well-structured in single layers of cuboidal or columnar cells. They are closely attached to surrounding cells by intercellular adhesion complexes. These cells also display an apicobasal polarity with a characteristic basal basement membrane that separates the epithelium from other tissues. In contrast, epithelial cells with a mesenchymal phenotype are characterized by the absence of polarity and intercellular adhesion junctions, hallmarks that have come to define EMT [45]. During the EMT process, cells lose the attachment of β-catenin and E-cadherin, which act to tightly link and attach surrounding epithelial cells together. This loss of attachment leads to a disruption of the adherens junctions [46]. These events then allow the mesenchymal phenotype to move freely and invade the surrounding extracellular matrix. In normal conditions, EMT provides a necessary function during embryogenesis, growth and wound healing. However, aberrant EMT can result in pathological conditions such as organ fibrosis and cancer metastasis. EMT mediated metastasis of malignant breast cancer epithelial cells can often form secondary tumors in the bone or lung [46]. EMT that occurs under normal conditions, such as embryogenesis, is referred to as type 1 EMT or classical EMT [47]. However, EMT that develops during inflammation, wound healing, tissue regeneration, and organ fibrosis is referred to as type 2 EMT, whereas EMT associated with cancer metastasis is termed type 3 EMT and plays

an important role in the development, growth and progression of breast cancer [47].

promote EMT [37, 43, 45].

Transcription factors also play a role in the initiation of EMT. Receptor activation by various growth factors, such as hepatocyte growth factor (HGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) are involved in the activation of various transcription factors involved in EMT. Growth factor-induced activation of transcription factors include zinc finger protein snail 1 (SNAIL1), (SNAIL2), zinc finger e-box-binding homeobox 1 (ZEB1), (ZEB2), twist, forkhead box protein 1 (FOXC1), (FOXC2), transcription factor 3 (TCF3), also known as (E47), and homeobox protein goosecoid (GSC) [37, 43, 45]. EMT also plays a role in the restructuring of extracellular matrix proteins by up-regulating fibronectin, collagen, proteases like MMPs, and other remodeling enzymes. In addition, autocrine and paracrine secretion of growth factors, cytokines, and extracellular proteins can modulate cancer cells phenotype and

Epigenetic modification, such as acetylation or methylation of the DNA, also can play a role in the EMT activation. For example, methylation of arginine (R531) by protein arginine methyltransferases 7 (PRMT7) plays a crucial role in inducing the EMT and the promotion of migratory and invasive behavior of breast cancer cells [48]. EMT can also be activated by expression of certain miRNAs, such as micro-RNA200/205 family (miRNA200) and (miRNA205), whose **Figure 3** shows the effects of γ-tocotrienol on the expression of EMT cellular biomarkers in the highly malignant MDA-MB-231 human breast cancer cells. Western blot analysis shows that MDA-MB-231 cells in the vehicle-treated control group displayed relatively low levels of expression for the epithelial cell markers cytokeratin 8, cytokeratin 18 and E-cadherin, and corresponding high levels of expression for the mesenchymal cell markers vimentin, fibronectin and total β-catenin (**Figure 3A**). Treatment with 3–7 μM γ-tocotrienol (MDA-MB-231) induced a dose-responsive reversal in epithelial versus mesenchymal cell marker expression (**Figure 3A**). Immunocytochemistry was then performed to confirm the finding in **Figure 3A**.

**Figure 3.** γ-Tocotrienol effects on epithelial versus mesenchymal cell markers expression. (A) Whole cell lysates were prepared from cells in each treatment group for subsequent separation by polyacrylamide gel electrophoresis (35 μg/lane) followed by western blot analysis. (B) Immunocytochemical analysis was done to confirm the finding shown in A. Cells in the various treatment groups were fixed, blocked, and incubated with specific primary antibodies for cytokeratin 8, cytokeratin 18, vimentin, and total level of β-catenin followed by incubation with Alexa Fluor 488-conjugated secondary antibody. Green staining in the photomicrographs (magnification 200×) indicates positive fluorescence staining for target proteins and the blue color represents counter staining of the cell nuclei with DAPI.

MDA-MB-231 cells in the vehicle-treated control group displayed a relatively low level of positive immunofluorescence staining for the epithelial cell marker cytokeratin 8, cytokeratin 18, and a relatively high level of positive immunofluorescence staining for the mesenchymal markers vimentin and β-catenin (**Figure 3B**). Treatment with 5 μM γ-tocotrienol resulted in a reversal of positive immunofluorescence staining of epithelial versus mesenchymal cell markers in MDA-MB-231 cells (**Figure 3B**) [14].

However, during conditions of Wnt activation, such as during wound healing, the pathway becomes acutely active during the healing process. During this time, Wnt ligands are translocated to the extracellular matrix where they bind to their receptor and co-receptors, which ultimately leads to phosphorylation of DVL. Phosphorylated DVL block GSK3β activity in the cytosolic complex. As a result, β-catenin will not be phosphorylated and no longer targeted for degradation. The stabilized β-catenin can now be translocated from the cytosol into the nucleus and induce transcription [61]. There are also numerous other proteins such as CREBbinding protein (CBP), polymerase associated factor 1 (PAF1), and Brahma (Brm), which work together as transcription factors to potentiate the Wnt signaling pathway [60]. Activation of this pathway leads to increase cyclin D1 expression, which is associated with cell cycle progression and growth. Similarly, an increase in myelocytomatosis (c-Myc) expression as a result of Wnt activation leads to increased cell proliferation and increase in MMP9 expression, which is involved in the disruption of the tight junctions [62]. An increase in snail and slug expression leads to a loss of the attachment of β-catenin and E-cadherin and the progression of EMT [63]. However, after the wound is healed and Wnt signaling is no longer needed, a negative feedback effect can occur by the action of certain proteins, such as DKK1 and axin 2, and represents highly controlled gene expression and cell growth [64]. However, cancer cells are characterized by an increased expression of Wnt ligands, as well as has numerous proteins in the cytosolic complex, such as β-catenin, APC, or axin 1, that can become mutated. These factors lead to the continuous activation of the Wnt pathway and is associated with increased

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**Figure 4** shows the effects of γ-tocotrienol treatment on the relative levels of signaling and regulatory proteins within the canonical Wnt and Hedgehog pathways. Total levels of the Wnt3a, FZD7 receptor, phosphorylated-LRP6 (active form), DVL2 and cyclin D1 were highly expressed in the vehicle-treated MDA-MB-231 cell line with corresponding relatively low expression of

**Figure 4.** Western blot analysis of γ-tocotrienol effects on the canonical Wnt and Hedgehog major regulatory proteins.

dish and maintained on serum-free defined media containing different doses of γ-tocotrienol over a 4-day culture period. Following treatment exposure, whole cell lysates were prepared from MDA-MB-231 in each treatment group for consequent separation by polyacrylamide gel electrophoresis (35 μg/lane) followed by western blot analysis for the major regulatory proteins of the Wnt pathway. (B) Whole cell lysates were prepared then subjected to polyacrylamide gel electrophoresis (30 μg/lane) and western blot analysis for detection of Shh ligand, PTCH2, Smo, GSK3β, Gli1 and

cells/100 mm

(A) Highly malignant MDA-MB-231 human breast cancer cells were initially seeded at density of 1 × 10<sup>6</sup>

tumor growth, motility, invasion and metastasis [7, 65].

SUFU levels within the Hedgehog pathway.
