Tocotrienols, a subgroup within the vitamin E family of compounds, have been shown to display potent anticancer activity and inhibit preneoplastic and neoplastic mammary epithelial cell proliferation at treatment doses that have little or no effect on normal cell growth and function. However, the specific intracellular mechanisms mediating the antiproliferative effects of tocotrienols are presently unknown. Because Akt and nuclear factor kappaB (NFkappaB) are intimately involved in mammary tumor cell proliferation and survival, studies were conducted to determine the effects of gamma-tocotrienol on Akt and NFkappaB activity in neoplastic +SA mammary epithelial cells in vitro. Treatment with 0-8 microM gamma-tocotrienol for 0-3 days caused a dose-responsive inhibition in +SA cell growth and mitotic activity, as determined by MTT colorimetric assay and proliferating cell nuclear antigen immunocytochemical staining, respectively. Studies also showed that treatment with 4 microM gamma-tocotrienol, a dose that inhibited +SA cell growth by more than 50% compared with that of untreated control cells, decreased intracellular levels of activated phosphotidylinositol 3-kinase-dependent kinase (PI3K)-dependent kinase 1 (phospho-PDK-1) and Akt, and reduced phospho-Akt kinase activity. Furthermore, these effects were not found to be associated with an increase in either phosphatase and tensin homologue deleted from chromosome 10 (PTEN) or protein phosphatase type 2A phosphatase activity. In addition, gamma-tocotrienol treatment was shown to decrease NFkappaB transcriptional activity, apparently by suppressing the activation of IkappaB-kinase-alpha/beta, an enzyme associated with inducing NFkappaB activation. In summary, these findings demonstrate that the antiproliferative effects of gamma-tocotrienol result, at least in part, from a reduction in Akt and NFkappaB activity in neoplastic +SA mammary epithelial cells.