**4. Canonical Wnt pathway**

The canonical Wnt pathway is one of the fundamental pathways that is overexpressed in cancer metastasis that is involved in the initiation of EMT [53, 54]. Wnt is an acronym derived from two proto-oncogene wingless and intel 1. At present, 19 Wnt ligands have currently been identified [55]. Those ligands form a large group of secreted glycoprotein that are secreted through autocrine and/or paracrine mechanisms. After DNA transcription and translation takes place, protein is translocated to the endoplasmic reticulum (ER), where a lipid tail is attached to the ligand by porcupine enzyme. Then, the ligand is transported to Golgi apparatus by Wntless/evenness enzymes. At the Golgi, a sugar moiety is linked to the ligand, facilitating its translocation to the ECM and binding to the receptors, respectively. Once the Wnt ligand is in the ECM, numerous proteins, such as dickkopf (DKK1), sclerostin (SOST), secreted frizzledrelated protein (SFRP), and Wnt inhibitory factor 1 (WIF1), play a role to block the signal. In contrast, other proteins, such as R-spondin (RSPO) and norrin can stimulate Wnt signaling [56]. Wnt pathway co-receptors are located in the lipid rafts which are microdomains in the cell membrane needed for the stimulation of signal transduction [7]. A number of proteins, such as glycosaminoglycan, dally and dly, are responsible for handling the Wnt ligand to the lipid rafts [55]. Ligand then bind to the co-receptors and induce activation of the signaling pathway [55].

The Wnt pathway can be stimulated canonically and non-canonically [57]. Nevertheless, the critical and most studied pathway is the canonical Wnt pathway, known to have a role in triggering EMT [58]. When there is no need for any developmental process, this pathway remains inactive and the receptor ligand is sequestered in the extracellular matrix by the action of number of specific binding proteins. Bound ligand to the Wnt receptor is inactive and prevents to a reduction in the phosphorylation of the disheveled protein (DVL), which is known to inhibit the cytosolic complex. The cytosolic complex composed of several proteins, such as glycogen synthase kinase 3 beta (GSK3β), axin 1, adenomatous polyposis coli (APC), and casein kinase 1 alpha (CK1α). The kinases in this complex remain active to phosphorylate the majority of the β-catenin, a biomarker for the canonical Wnt pathway activation. Phosphorylated β-catenin is then targeted for degradation by proteasomal enzymes [59]. In the nucleus, β-catenin is translocated out of the nucleus by the action of APC, Ran, and Manchette-associated binding adaptor protein 3 (BP3). The T-cell factor/lymphoid enhancer factor (LEF/TCF) area in the DNA is the binding location of β-catenin and is hidden by Groucho, histone deacetylase (HDAC), and glucose transporter-binding protein (GtBP) as a mechanism to rid the cell of β-catenin activity. Finally, the rest of β-catenin in the nucleus is sequestered by Chibby (CBY) and inhibitor of β-catenin and TCF 4 (ICAT) [60]. The summation of these events results in the blockade of Wnt signaling and downstream gene expression and mitogenesis.

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

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 mark-

The canonical Wnt pathway is one of the fundamental pathways that is overexpressed in cancer metastasis that is involved in the initiation of EMT [53, 54]. Wnt is an acronym derived from two proto-oncogene wingless and intel 1. At present, 19 Wnt ligands have currently been identified [55]. Those ligands form a large group of secreted glycoprotein that are secreted through autocrine and/or paracrine mechanisms. After DNA transcription and translation takes place, protein is translocated to the endoplasmic reticulum (ER), where a lipid tail is attached to the ligand by porcupine enzyme. Then, the ligand is transported to Golgi apparatus by Wntless/evenness enzymes. At the Golgi, a sugar moiety is linked to the ligand, facilitating its translocation to the ECM and binding to the receptors, respectively. Once the Wnt ligand is in the ECM, numerous proteins, such as dickkopf (DKK1), sclerostin (SOST), secreted frizzledrelated protein (SFRP), and Wnt inhibitory factor 1 (WIF1), play a role to block the signal. In contrast, other proteins, such as R-spondin (RSPO) and norrin can stimulate Wnt signaling [56]. Wnt pathway co-receptors are located in the lipid rafts which are microdomains in the cell membrane needed for the stimulation of signal transduction [7]. A number of proteins, such as glycosaminoglycan, dally and dly, are responsible for handling the Wnt ligand to the lipid rafts [55]. Ligand then bind to the co-receptors and induce activation of the signaling pathway [55]. The Wnt pathway can be stimulated canonically and non-canonically [57]. Nevertheless, the critical and most studied pathway is the canonical Wnt pathway, known to have a role in triggering EMT [58]. When there is no need for any developmental process, this pathway remains inactive and the receptor ligand is sequestered in the extracellular matrix by the action of number of specific binding proteins. Bound ligand to the Wnt receptor is inactive and prevents to a reduction in the phosphorylation of the disheveled protein (DVL), which is known to inhibit the cytosolic complex. The cytosolic complex composed of several proteins, such as glycogen synthase kinase 3 beta (GSK3β), axin 1, adenomatous polyposis coli (APC), and casein kinase 1 alpha (CK1α). The kinases in this complex remain active to phosphorylate the majority of the β-catenin, a biomarker for the canonical Wnt pathway activation. Phosphorylated β-catenin is then targeted for degradation by proteasomal enzymes [59]. In the nucleus, β-catenin is translocated out of the nucleus by the action of APC, Ran, and Manchette-associated binding adaptor protein 3 (BP3). The T-cell factor/lymphoid enhancer factor (LEF/TCF) area in the DNA is the binding location of β-catenin and is hidden by Groucho, histone deacetylase (HDAC), and glucose transporter-binding protein (GtBP) as a mechanism to rid the cell of β-catenin activity. Finally, the rest of β-catenin in the nucleus is sequestered by Chibby (CBY) and inhibitor of β-catenin and TCF 4 (ICAT) [60]. The summation of these events results in the

blockade of Wnt signaling and downstream gene expression and mitogenesis.

ers in MDA-MB-231 cells (**Figure 3B**) [14].

**4. Canonical Wnt pathway**

90 Vitamin E in Health and Disease

**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. (A) Highly malignant MDA-MB-231 human breast cancer cells were initially seeded at density of 1 × 10<sup>6</sup> cells/100 mm 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 SUFU levels within the Hedgehog pathway.

Naked 1 (a negative regulator of Wnt pathway (**Figure 4A**). Treatment with 3–7μM γ-tocotrienol (MDA-MB-231) induced a dose-dependent decline in Wnt3a, FZD7 receptor, phosphorylated-LRP6, DVL2, cyclin D1 levels, and a corresponding increase in Naked 1 level as compared to cells in their respective vehicle-treated control groups (**Figure 4A**). These findings indicate that γ-tocotrienol inhibition of EMT is mediated in part by a suppression of canonical Wnt signaling. Similar results were observed in T47-D breast cancer line (data not shown). Previous studies have shown that inhibition of Wnt signaling resulted in a reduction in nuclear factor erythroid 2-related factor 2 (Nrf2) activity, a transcription factor associated with the promotion of EMT [66–68]. At present, it is not known if γ-tocotrienol reversal of EMT involves a corresponding decrease in Nrf2 activity. Additional studies are required to determine if Nrf2 plays a role in the anticancer effects of γ-tocotrienol. In summary, experimental evidence strongly suggests that γ-tocotrienol therapy may provide therapeutic value in the treatment of highly malignant breast cancer that is characterized by aberrant canonical Wnt signaling.

N-terminal of the signaling piece, leading to an increase in its hydrophobicity, localization and binding to the receptor [73]. The canonical Hedgehog signaling pathway has several vital components which play a role in modulating signal intensity. Most of the components within the Hedgehog pathway include the Hedgehog ligand, the PTCH receptor, Smo, and the cytosolic complex and downstream effectors, which consist of suppressor of fused (SUFU) and Gli family of proteins. The Gli family is an important component of the Hedgehog pathway which is divided into three forms known as Gli1, Gli2, and Gli3. Gli transcription factors can activate the signal, have dual function to stimulate or impede the signal [79]. A number of kinases, such as GSK3β, CK1α, and protein kinase A, are known to be essential in the regulation of Hedgehog signaling [80]. The PTCH receptor of this pathway is located in the lipid raft

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Activation of the Hedgehog pathway can be blocked in the absence of ligand expression or a lack of mutation in PTCH and/or Smo [79]. In such cases, the inhibitory effect of PTCH on Smo is intact and Hedgehog ligand transport to the cell membrane is prevented and receptor activation and signal transduction does not occur [79]. In contrast, activation of the Hedgehog pathway will result in conditions when the Hedgehog ligand is highly expressed, and/or when mutation of PTCH and/or Smo occurs [79]. In these conditions, inhibitory effect of PTCH on Smo is absence and Smo can freely travel to the cell membrane, leading to the phosphorylation of SUFU and the transcription factor in the cytosolic complex. Once this occurs, Gli separates from the cytosolic complex proteins and then translocates into the nucleus where it promotes an increase in the Hedgehog target gene expression [79]. Recent studies have shown a direct connection between EMT and stemness of breast cancer resulting that is directly associated with the activation of the canonical Hedgehog signaling and the development of tumor

**Figure 4B** shows the effects of γ-tocotrienol treatment on signaling protein levels and activation within the Hedgehog pathways. Results show that the Hedgehog Shh ligand is relatively high in MDA-MB-231 breast cancer cells in the vehicle-treated control group. Similarly, PTCH2 receptor, Smo, GSK3β, and Gli1 were highly expressed, while the inhibitor for Hedgehog signaling SUFU displayed a relatively low level of expression in the vehicle-treated MDA-MB-231 human breast cancer cells (**Figure 4B**). Treatment with γ-tocotrienol induced a dose-dependent decrease in Shh ligand expression, as well as a dose-responsive reduction in PTCH2 receptor, Smo, GSK3β, and Gli1, and a corresponding increase in SUFU protein levels, as compared to MDA-MB-231 cells in the vehicletreated control group (**Figure 4B**). These data indicated that γ-tocotrienol inhibition of EMT is also mediated by a suppression of canonical Hedgehog pathway and provides further evidence that γ-tocotrienol treatment may provide significant benefit in the treat-

Results from these reports show that treatment with 0–5 μM γ-tocotrienol induced a significant dose-dependent inhibition of highly malignant MDA-AM-231 human breast cancer

microdomains of the plasma membrane [8].

recurrence and metastasis [71].

ment of metastatic breast cancer.

**6. Conclusion**
