Amit B. Shirode and Paul W. Sylvester
Aim: To characterize the intracellular signaling mechanisms mediating the synergistic anticancer effects of combined ?-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 ? -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 ?-tocotrienol or celecoxib alone inhibited Akt activation and downstream signaling and NF?B activation. Similar treatment with ?-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 ?-tocotrienol and celecoxib resulted in a large decrease ErbB2-4 receptor levels and activation, a decrease in PGE2 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 ?-tocotrienol and celecoxib treatment in +SA mammary tumor cells are mediated by COX-2-dependent mechanisms associated with a suppression in PGE2 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 NF?B mitogenic signaling.
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