Mechanisms mediating antiproliferative effects of combined treatment of gamma-tocotrienol with ppargamma agonists and antagonists in mammary cancer cell proliferation

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand activated transcription factor that regulates lipid metabolism and adipocyte differentiation. Experimental evidence shows that PPARgamma is over expressed in breast cancer and tissues, which may lead to increased production of lipogenic enzymes that help in the growth of highly proliferating cancer cells. Therefore, inhibition of PPARgamma expression and/or activity may be beneficial in the treatment of breast cancer. gamma-Tocotrienol is a naturally occurring isoform of vitamin E family of compounds that displays potent anticancer activity. Studies have shown that gamma-tocotrienol stimulates endogenous PPARgamma activity. Therefore, it was hypothesized that combined treatment of gamma-tocotrienol with PPARgamma agonists or antagonists may show anticancer effects in mammary cancer cells. In addition experiments were conducted to determine whether these effects are mediated via PPARgamma-dependent or -independent pathway. Results showed that while treatment alone with gamma-tocotrienol, PPARgamma agonists (rosiglitazone or troglitazone) or PPARgamma antagonists (GW9662 or T0070907) resulted in a dose-responsive inhibition of mammary cancer cell proliferation. Combined treatment of gamma-tocotrienol with PPARgamma agonists reversed the growth inhibitory effects of gamma-tocotrienol, whereas combined treatment of gamma-tocotrienol with PPARgamma antagonists synergistically inhibited mammary cell growth. In addition, combined treatment of gamma-tocotrienol and PPARgamma agonists caused an increase in PPARgamma expression and PPRE reporter activity. In contrast, combined treatment of gamma-tocotrienol with PPARgamma antagonists resulted in a decrease in PPARgamma expression and activity, and a corresponding decrease in PI3K/Akt mitogenic signaling in breast cancer cells. These results showed that combined treatment of gamma-tocotrienol with PPARgamma antagonists mediate anticancer effects in part by PPARgamma-dependent pathway. Further, studies were conducted to determine effect of combined treatment of gamma-tocotrienol with PPARgamma antagonists on adipogenic factors. Results showed that this combination treatment suppressed adipogenic transcription factors, C/EBP and SREBP-1c, and their target lipogenic enzymes. However, PPARgamma silenced studies showed that these effects were mediated by PPARgamma-independent pathway. Furthermore experiments were conducted on PPARgamma negative +SA cells to confirm if the anticancer effects of combination treatment are mediated though PPARgamma-independent pathway. Results showed that combined treatment of gamma-tocotrienol and PPARgamma antagonist treatments was associated with a reduction in COX-2 and PGDS expression and corresponding decrease in PGD2 synthesis independent of PPARgamma. Therefore, although the initial findings showed that the combined treatment of gamma-tocotrienol with PPARgamma antagonists was mediated in part through PPARgamma-dependent pathway, later experiments confirmed that the antiproliferative effects of this combination treatment are mediated by PPARgamma-independent pathway. Overall, these results emphasize the benefit of this combination treatment against breast cancer.