Figure 4

α-TEA alone and in combination with TAM induces biomarkers of endoplasmic reticulum stress, and siRNA knockdowns show necessary roles for CHOP, DR5, and JNK. (a) Western immunoblot analyses, using aliquots of cell lysates from cells treated with 20 μM α-TEA and 1 μM TAM in Figure 3b, were performed to assess pJNK2/1, CHOP, and DR5 (L/S) protein levels, as well as endoplasmic reticulum stress marker GRP78 protein expression with GAPDH as loading control. (b) MCF-7/TAMR cells transfected with siRNAs to CHOP, DR5, and JNK as well as control siRNA (labeled Control) were treated with TAM (1 μM) + α-TEA (20 μM) for 1 day. Western immunoblot analyses were performed to determine degree of apoptosis, as measured by cleaved PARP (cPARP), pJNK2/1, CHOP, and DR5 (L/S) protein levels, with GAPDH serving as the loading control. Data from (a) and (b) are representative of three individual experiments. CHOP, Ccaat-enhancer-binding protein (C/EBP) homologous protein; DR5 (L/S), death receptor 5 long/short; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP78, glucose-regulated protein-78; JNK, c-Jun N-terminal kinase; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; PARP, poly (ADP-ribose) polymerase; siRNA, small interfering RNA; TAM, tamoxifen; TAMR, tamoxifen resistant; α-TEA, RRR-α-tocopherol ether-linked acetic acid analogue.