α-TEA decreased Bcl-2 and c-FLIP (L) protein levels via caspase-8 dependent ER stress-mediated JNK/CHOP/DR5 pathway.

A. MDA-MB-231 and MCF-7 cells were treated with 40 µM α-TEA for 9, 15, and 24 hrs. Western blot analyses were performed to evaluate Bcl-2 and c-FLIP (L) protein levels with GADPH as loading control. B. MCF-7 cells were transiently transfected with JNK, CHOP, and DR5 siRNA or control siRNA followed by treatment with 40 µM α-TEA or vehicle for 18 hrs. Bcl-2 and c-FLIP (L) protein levels were determined by western blot analyses, using GAPDH as loading control. C. MDA-MB-231 and MCF-7 cells were cultured with ER-stress inhibitor salubrinal at 40 µM or caspase-8 inhibitor (Z-IETD-FMK) at 2 µM plus 40 µM α-TEA for 18 hrs. Bcl-2 and c-FLIP (L) protein levels were determined by western blot analyses, using GAPDH as loading control. D. MCF-7 cells were transiently transfected with Itch siRNA or control siRNA followed by treatment with 40 µM α-TEA or vehicle for 18 hrs. c-FLIP (L) protein levels were determined by western blot analyses. E. MDA-MB-231 and MCF-7 cells were transiently transfected with siRNA to itch using non-specific siRNA as negative control followed by treatment with 40 µM α-TEA for 18 hrs. Apoptosis was determined by Annexin V/FACS. F. MDA-MB-231 and MCF-7 cells were treated with 40 µM α-TEA for 9, 15, and 24 hrs. mRNA levels of Bcl-2 were determined by RT-PCR G. MCF-7 cells were transiently transfected with wild-type c-FLIP plasmid or vector control followed by treatment with 40 µM α-TEA for 18 hrs. Western blot analyses were performed to evaluate over expression of c-FLIP on ability of α-TEA to cleave PARP and caspase-8, and down-regulate c-FLIP (L), pJNK2/1, CHOP, DR5 (L/S), peIF-2α and GRP78 protein expression using GAPDH as loading control. H. MCF-7 cells were transiently transfected with siRNA to Itch, using non-specific siRNA as negative control followed by treatment with 40 µM α-TEA for 18 hrs. Western blot analyses were performed to evaluate elevated levels of c-FLIP (L) on ability of α-TEA to cleave PARP and caspase-8 and to down-regulate pJNK2/1, CHOP, DR5 (L/S) and GRP78 protein levels using GAPDH as loading control. Data for A, B, C, D, F, G and H are representative of two or more individual experiments. Data from E are the mean ± S.D. of three independent experiments. * p<0.05 = significantly different from control siRNA determined by t-test.