a TEA chroman 6 yloxyacetic acid, known as RRR a tocopherol ether linked acetic acid analog or RRR a toco pheryloxyacetic acid is a nonhydrolyzable ether analog of RRR a tocopherol. a TEA has been shown to be a potent pro apoptotic agent both in vitro and in vivo animal study in breast, prostate and ovarian cancer cells. Recently, a TEA has been shown to delay tumor onset and to inhi bit the progression and metastatic spread in a clinically relevant Inhibitors,Modulators,Libraries model of spontaneous mammary cancer, further highlighting the translational potential of this anticancer agent. Mechanisms involved in a TEA induced apop tosis include activation of JNK c Jun, p73 NOXA and Fas death receptor 5, and suppression of c FLIP L, sur vivin and phospho Akt leading to death receptor mediated caspase 8 activation and mitochondria depen dent apoptosis.

Data presented here show that a TEA in combination with DOXO or CDDP significantly enhances apoptosis of p53 mutant, triple negative human breast cancer cells Inhibitors,Modulators,Libraries by targeting p73 mediated p53 dependent pro apoptotic and anti apoptotic genes via c Abl, JNK and Yap signal ing pathways. Materials and methods Chemicals a TEA was made in house as previously described. DOXO and CDDP were purchased from Sigma. Phosphoinositide 3 kinase inhibitor was purchased from Cell Signaling Tech nology. Cell culture p53 mutant, triple negative human breast cancer cell lines MDA MB 231, BT 20 and MDA MB 468 were purchased from the American Type Culture Collection. MDA MB 231 and BT 20 cells were cultured in MEM media with 10% FBS, and Inhibitors,Modulators,Libraries MDA MB 468 cells were cultured in Dulbeccos MEM media with 10% FBS.

All three p53 mutant TNBC cell lines used in these studies were originally obtained from human samples so no isogenic counterparts expressing wildtype p53, ER and progesterone receptor are available for use as controls. For experiments, FBS was reduced to 2% to better mimic low in vivo serum exposure and cells were allowed to attach overnight before Inhibitors,Modulators,Libraries treatment. a TEA was dissolved in ethanol Inhibitors,Modulators,Libraries as a stock solution. Concentrations of ethanol used in vehicle treatments were 0. 025 to 0. 05% to match the ethanol content in the different final concentrations of a TEA treatments. DOXO and CDDP were dissolved in H2O. Quantification of apoptosis Apoptosis was quantified by annexin V FITC PI assays following the manufacturers instructions.

Fluorescence was measured using fluorescence activated cell sorter analyses with a FACSCalibur flow cytometer, and data were analyzed using CellQuest software. Cells displaying phosphatidylserine on their surface were considered sellekchem apoptotic. Nuclear and cytoplasmic fractionation Cytoplasmic and nuclear fractions were prepared as pre viously described. Briefly, whole cell lysates were centrifuged to obtain supernatant and pellet. The super natant was centrifuged again and the resulting superna tant was used as the cytosolic fraction.