**6. Conclusion**

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

The canonical Hedgehog pathway is characteristically over active in many forms of metastatic breast cancer and is associated with enhanced migration, invasion, stemness and self-renewal of cancer cells [69–72]. Over activity of the Hedgehog pathway is also associated with playing a role in promoting EMT [71]. The Hedgehog ligand was first discovered in the *Drosophila* fruit fly [73]. Several human Hedgehog ligands have also been identified that are involved in cell growth and controlled organ formation by insuring that the tissue reaches the accurate size and position. In the adult, this pathway normally remains quiescent. However, activation of the Hedgehog pathway may be triggered during tissue maintenance and regeneration [74]. A link between Hedgehog signaling and developmental defects was first discovered in 1996, and later that year a link between Hedgehog signaling and cancer was found when the tumor suppressor gene patched (PTCH) was discovered [67]. Soon afterwards, the Hedgehog cell service signaling transducer, smoothened (Smo), was discovered and found to have the potential to function as an oncogene. These findings lead to the development of the Hedgehog pathway inhibitor, cyclopamine, and successful clinical trials using cyclopamine and similar agents followed [75]. Hedgehog ligands are produced by three different genes. The first gene is the Indian Hedgehog (Ihh) and is found in gut, skeletal muscle, and chondrocytes [76]. The

breast cancer that is characterized by aberrant canonical Wnt signaling.

second gene is the Desert Hedgehog (Dhh) and is expressed in the testis [76].

The third gene is the Sonic Hedgehog (Shh) and is involved in many developmental processes [76]. Shh is called a morphogen since the signal of this ligand relies on its concentration [77]. The Shh is produced from zone of polarizing activity (ZPA), which is located on the posterior side of the limb bud in the embryo [78]. The Hedgehog pathway has a link with the formation of specific types of humans cancer [74]. After the transcriptional and translational process occur, this ligand is secreted as a precursor protein, and the ligand is subsequently subjected to several post translational modifications [73]. Autocatalytic cleavage then splits the ligand into two parts. One part is the signaling molecule, while the other part appears to have no function. A cholesterol molecule and palmitic acid moiety are then added to C-terminal and

**5. Canonical Hedgehog pathway**

92 Vitamin E in Health and Disease

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 cell growth after a 4-day culture period. Furthermore, canonical Wnt and Hedgehog signaling are highly expressed in these triple negative breast cancer cells, and γ-tocotrienol growth inhibitory effects are associated with a reduction in Wnt and Hedgehog signaling and regulatory proteins. Since γ-tocotrienol also induces a reversal of EMT in these cells and canonical Wnt and Hedgehog signaling pathways are involved in promoting EMT, it can be concluded that γ-tocotrienol inhibition of EMT is mediated by a corresponding reduction in canonical Wnt and Hedgehog signaling in malignant MDA-MB-231 human breast cancer cells. This hypothesis is further evidenced by the finding that γ-tocotrienol inhibition of Wnt and Hedgehog signaling and reversal of EMT is associated with a significant decrease in migration, invasion and stemness of these cells [12].

**Acknowledgements**

**Conflict of interest**

**Author details**

**References**

There is no conflict of interest.

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Rayan Ahmed and Paul W. Sylvester\*

\*Address all correspondence to: sylvester@ulm.edu

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College of Pharmacy, University of Louisiana at Monroe, Monroe, LA, USA

Medicine. 2015;**5**(4):pii: a006098. DOI: 10.1101/cshperspect.a006098

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[2] DeSantis C et al. Breast cancer statistics, 2013. CA: A Cancer Journal for Clinicians.

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[5] Pike LJ. Lipid rafts: Bringing order to chaos. Journal of Lipid Research. 2003;**44**(4):655-667 [6] Pike LJ. The challenge of lipid rafts. Journal of Lipid Research. 2009;**50**(Suppl):S323-S328 [7] Feng Q, Gao N. Keeping Wnt signalosome in check by vesicular traffic. Journal of

[8] Karpen HE et al. The sonic hedgehog receptor patched associates with caveolin-1 in cholesterol-rich microdomains of the plasma membrane. The Journal of Biological Che-

The work was performed in College of Pharmacy, University of Louisiana at Monroe, Monroe, LA. This work was supported by grants from First Tech International Ltd. (Hong Kong) and the Louisiana Cancer Foundation. The authors thank the First Tech International Ltd. for generously providing γ-tocotrienol for use in these studies. The authors also thank Dr. Karen P. Briski for her generous technical assistance and use of the laser confocal microscope.

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

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

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Metastasis is still the primary cause for the mortality (90%) in cancer patients with cancer. While a great deal of progress has been recently achieved in the further understanding of the molecular and cellular mechanisms involved in the metastatic process, these mechanisms are not completing understood and clinical therapies for the management and treatment of metastatic cancer remains insufficient. Expanding knowledge in gene expression, cellular behavior, and biological events of cancer cells will provide important and novel insights for the treatment of metastatic breast cancer. New biomarkers in areas, such as EMT will provide innovative chances in predictive methods of the metastatic potential of a primary tumor and a novel target for therapy. Experimental results summarized in **Figure 5** indicates some of the key targets in the treatment of EMT and metastasis and the possible role of γ-tocotrienol in the prevention and treatment of these processes.

In summary, experimental evidence demonstrates that γ-tocotrienol reversal of EMT results, at least in part, through the inhibition of canonical Wnt and Hedgehog signaling. These findings also suggest that supplemental treatment with γ-tocotrienol may be effective in providing significant benefit in the prevention and treatment of metastatic breast cancer.

**Figure 5.** Schematic representation of γ-tocotrienol effects on the canonical Wnt and Hedgehog pathways and EMT. γ-Tocotrienol inhibits Wnt signaling by decreasing the expression of Wnt3a ligand, FZD7/LRP6 complex activation, DVL2 and cyclin D1 and a corresponding increase in Naked 1 level. Additionally, γ-tocotrienol inhibits Hedgehog signaling by decreasing the expression of Shh ligand, PTCH2, Smo, GSK3β, and Gli1 associated with a corresponding increase in SUFU levels. Several other cytosolic and nuclear proteins were minimized which can ultimately lead to a suppression in gene expression associated with EMT.
