Elizabeth extremely lipophilic tocotrienol was suspended in an answer of sterile one hundred thousand BSA as described previously. PI3K/Akt mitogenic signaling in these breast cancer cell lines, and treatment ubiquitin-conjugating effects were evaluated by additional studies on the expression of PPAR and PPAR coactivators. from these studies provides insights as to potential benefits of these therapies in the treatment of breast cancer, and more characterize the anticancer mechanism of action of tocotrienol, as well as PPAR agonist and antagonists. All reagents were purchased from Sigma Chemical Company unless otherwise stated. Pure tocotrienol was generously provided as a gift by First Tech International Ltd. e PPAR agonists, troglitazone and rosiglitazone, and the PPAR antagonists, GW9662 and T0070907, were acquired from Cayman Chemicals. Fetal bovine serum was purchased from American Type Culture Collection. Antibodies for actin, PPAR, Akt, phospho Akt, PTEN, phospho PTEN, PDK 1, PI3K, pyrazine cleaved caspase 3, and cleaved PARP were purchased from Cell Signaling Technology. Antibodies for RXR, CBP C 20, SRC 1, and CBP p/300 were purchased from Santa Cruz Biotechnology. Goat antirabbit and anti mouse secondary antibodies were obtained from PerkinElmer Biosciences. 2. 2. Cell Lines and Culture Conditions. e estrogen receptor bad MDA MB 231, and the estrogen receptor positive MCF 7 breast carcinoma cell lines were obtained from American Type Culture Collection. MDAMB 231 and MCF 7 breast cancer cells were cultured in modi fied Dulbeccofis modified Eagle Medium /F12 supplemented with ten percent fetal bovine serum, 10 g/mL insulin, 100 U/mL penicillin, 0. 1 mg/mL streptomycin at 37 C in a atmosphere of 95% air and 50-percent CO2 in a humidified incubator. For subculturing, cells were rinsed twice with sterile Ca2 and Mg2 free phosphate buffered saline and incubated in 0. 05-20 trypsin containing 0. 025% EDTA in PBS for 5 min at 37 C. e produced cells were centrifuged, re-suspended in serum containing media, and counted using a hemocytometer. Briefiy, an appropriate number of tocotrienol was incubated over night at 37 C with constant shaking, then added to a little level of sterile 10 % BSA in water and first dissolved in 100 L of 100% ethanol. hedgehog antagonist is stock solution was then used to get ready various concentrations of treatment media. Stock solutions of rosiglitazone, troglitazone, GW9662 and T0070907 were prepared in DMSO. Ethanol and/or DMSO was added to all treatment media such that the ultimate concentration was the same in all treatment groups within any given experiment and was always less than 2. 4. Development Studies.