Synergistic antiproliferative effects of combined gamma-tocotrienol and celecoxib treatment on neoplastic mouse mammary epithelial cells

The selective cyclooxygenase COX-2 inhibitor, celecoxib, and the vitamin E isoform, gamma-tocotrienol, both display potent anticancer activity. However, high dose clinical use of selective COX-2 inhibitors has been limited by gastrointestinal and cardiovascular toxicity, whereas limited absorption and transport of gamma-tocotrienol by the body has made it difficult to obtain and sustain therapeutic levels in the blood and target tissues. Studies were conducted to characterize the synergistic anticancer antiproliferative effects of combined low dose celecoxib and at higher doses gamma-tocotrienol treatment on mammary tumor cells in culture. Combined treatment with subeffective doses of gamma-tocotrienol (0.25 muM) and celecoxib (2.5 muM) resulted in a synergistic antiproliferative effect, as determined by isobologram analysis. This growth inhibitory effect was associated with a reduction in PGE 2 synthesis, and decrease in COX-2, phospho-Akt and phospho-NFkappaB levels. Further investigation showed that both drugs inhibited the activation of PI3K/PDK/Akt mitogenic signaling pathway and NFkappaB transcriptional activation by decreasing the phosphorylation Akt, GSK-3beta, NFkappaB, IKK-alpha/beta and IkappaB-alpha. While gamma-tocotrienol down-regulated the expression and activation of EGF receptors at higher doses, celecoxib at higher doses only inhibited the receptor activation without any effect on level of EGF receptor expression. However, combined synergistic treatment caused inhibition of EGF receptor activation at subeffective doses. The synergistic treatment caused up-regulation of EP2 and EP4 prostaglandin receptors as a counter-regulatory response to decreased PGE2 levels. Combined treatment also resulted in up-regulation of prostaglandin catabolizing enzyme PGDH, leading to further deactivation of PGE2. These studies have demonstrated that the synergistic antiproliferative effects of gamma-tocotrienol and celecoxib in mammary tumor cells are mediated by several mechanisms dependent and independent of COX-2; COX-2-independent mechanisms involve simultaneous inhibition of activation of PI3K/PDK/Akt mitogenic signaling pathway and NFkappaB transcription pathway, and down-regulation of EGF receptor activation. COX-2-dependent mechanisms involve inhibition of transactivation of EGF receptors by PGE 2 and the increased inactivation of intracellular PGE2 by prostaglandin dehydrogenase enzymes. These findings also suggest that combination therapy with these agents may provide enhanced therapeutic response in breast cancer patients, while avoiding the toxicity associated with high-dose COX-2 inhibitor monotherapy and overcoming the problem of limited bioavailability of gamma-tocotrienol at higher doses.