| Antiproliferative Effects of gamma-Tocotrienol are Associated with Suppression of c-Myc Expression and Aerobic Glycolysis in Mammary Cancer Cells (Major Professor: Paul W. Sylvester, Ph.D.) c-Myc is a helix-loop-helix leucine zipper transcription factor that plays a central role in regulating numerous cellular functions including cell proliferation, differentiation, growth, and apoptosis. gamma-Tocotrienol is a member within the vitamin E family of compounds that displays potent antiproliferative and apoptotic activity against a wide range of cancer cell types at a treatment doses that have little or no effect on normal cell viability. Studies were conducted to determine the effects of gamma-tocotrienol on oncogenic c-Myc activity and metabolic degradation in mouse +SA and human MCF-7 mammary cancer cells. Treatment with 0-411M (+SA) or 0-811M (MCF-7) y-tocotrienol resulted in a dose-responsive inhibition of mammary tumor cell growth, and these effects associated with a decrease in total c-Myc, phosphorylated-S62-c-Myc, and a corresponding increase in p-T58-c-Myc. gamma-Tocotrienol treatment also caused a decrease in PI3K, phosphorylated (activated) Akt, phosphorylated (inactive) GSK-30, phosphorylated (activated) mTOR, and phosphorylated(activated) Erk 1/2 levels, but had no effect on MYC mRNA levels in these cells. These antiproliferative effects were also associated with a decrease in cyclin Dl and cyclin dependent kinase 4 (CDK4), and corresponding increase in p27. Additional studies showed that similar treatment with gamma-tocotrienol resulted in an increase in FBW7 levels, an E3 ligase that initiates ubiquitination of c-Myc, but had no effect on protein phosphatase 2A (PP2A) and Pin 1 propyl isomerase levels in these tumor cells. gamma-Tocotrienol induced c-Myc degradation was also found to be associated with GSK-3beta activation and proteasomal function. Furthermore, cancer cells display increased glucose consumption and a corresponding increase in lactate production even in presence of oxygen as compared to the normal cells and this process is called as aerobic glycolysis. Aerobic glycolysis is regulated by activation of the PI3K/Akt/mTOR pathway and oncogenic transcription factors such as, c-Myc and the hypoxia inducible factor, HIF-1alpha. Therefore, additional studies were conducted to determine the effects of gamma-tocotrienol on aerobic glycol ysis in mouse +SA and human MCF-7 breast cancer cells. Treatment with 0-4microM (+SA) or 0-10microM (MCF-7) gamma-tocotrienol resulted in a dose-responsive inhibition of mammary tumor cell growth. Treatment with antiproliferative doses of gamma-tocotrienol was also found to result in a relatively large reduction in glucose utilization and a corresponding decrease in intracellular ATP production and extracellular lactate excretion by +SA and MCF-7 mammary tumor cells. Furthermore, Western blot analysis showed that these anticancer effects were also associated with a large decrease in the expression of intracellular enzymes involved in regulating aerobic glycolysis including hexokinase-II, phosphofructokinase, pyruvate kinase M2, and lactate dehydrogenase A. In addition, these gamma-tocotrienol-induced effects were associated with a corresponding reduction in signaling proteins associated with regulating aerobic glycolysis, particularly phosphorylated (active) Akt, phosphorylated (active) mTOR, and c-Myc, but had no effect on the protein expression of HIF-lalpha or GLUT-1. In summary, these findings demonstrate that the antiproliferative effects of 7-tocotrienol are mediated, at least in part, by a reduction in oncogenic c-Myc (S62) levels, PI3K/Akt/mTOR and RAS/MEK/MAPK mitogenic signaling and a corresponding increase in GSK-3betadependent ubiquitination and degradation of c-Myc. Furthermore, these results demonstrate that the antiproliferative effects of y-tocotrienol are also associated with concurrent inhibition of aerobic glycolysis and PI3K/Akt/mTOR and c-Myc signaling. Taken together, these findings suggest that gamma-tocotrienol treatment might be beneficial in breast cancer with aberrant c-Myc signaling and altered metabolic changes. |
