AIM: To characterize the intracellular signaling mechanisms mediating the synergistic anticancer effects of combined γ-tocotrienol and celecoxib treatment in neoplastic +SA mouse mammary epithelial cells in vitro. METHODS: +SA mammary tumor cells in different treatment groups were maintained in serum-free defined media containing 10ng/ml EGF as a mitogen and exposed to various doses of γ-tocotrienol and celecoxib alone or in combination. After a 96 hr culture period, cells were collected and whole cell lysates were subjected to Western blot analysis to determine treatment effects on intracellular signaling proteins associated with EGF-dependent mitogenesis and survival, and prostaglandin synthesis and responsiveness. RESULTS: Treatment with high doses of γ-tocotrienol or celecoxib alone inhibited Akt activation and downstream signaling and NFκB activation. Similar treatment with γ-tocotrienol also decreased concentration and activation of ErbB2-4 receptors, whereas celecoxib only inhibited ErbB2-4 receptor activation. In contrast, combined treatment with subeffective doses of γ-tocotrienol and celecoxib resulted in a large decrease ErbB2-4 receptor levels and activation, a decrease in PGE(2) levels, and a corresponding increase in prostaglandin EP2 and EP4 receptor levels. Combined treatment also induced an increase in the prostaglandin catabolizing enzyme, PGDH. CONCLUSION: The synergistic anticancer effects of combined low dose γ-tocotrienol and celecoxib treatment in +SA mammary tumor cells are mediated by COX-2-dependent mechanisms associated with a suppression in PGE(2) levels, as well as, COX-2-independent mechanisms associated with a reduction in ErbB2-4 receptor levels, activation, and subsequent reduction in downstream Akt and NFκB mitogenic signaling.