it demonstrate that celecoxib induces apoptosis in non-small cell lung cancer cell lines involving the activation of the extrinsic demise receptor pathway through both DR5 induction and h FLIP down-regulation. We have found that celecoxib downregulates d FLIP through Gemcitabine 122111-03-9 facilitating ubiquitin/ proteasome dependent protein degradation. Nevertheless, the signaling process resulting in celecoxib caused h FLIP destruction is not known. In an attempt to show the mechanism underlying celecoxib caused c FLIP degradation, we have revealed a novel mechanism of c FLIP degradation through GSK3 inhibition. To the most useful of our knowledge, that is the first study demonstrating a linkage between GSK3 inhibition and c FLIP downregulation, thus highlighting a new mechanism through which GSK3 modulates the extrinsic apoptotic pathway. Celecoxib, antibodies and dimethy celecoxib against caspases and DR5 were the same as described previously. Human recombinant TRAIL was bought from PeproTech, Inc. LY294002 and rapamycin were obtained from LKT Laboratories, Inc. Page1=46 31 8220 and wortmannin Latin extispicium were obtained from Biomol. MG132, LiCl, SB216763 and SB415286 were purchased from Sigma Chemicals. G 6979, GF109203X, g 6983 and rottlerin were bought from EMD Calbiochem. Rabbit polyclonal antibodies against p Akt, p GSK3B, p GSK3/B, and p S6 were purchased from Cell Signaling Technology, Inc.. Rabbit polyclonal antibodies against p and GSK3/B FOXO3 were purchased from Upstate. Mouse monoclonal anti FLIP antibody was obtained from Alexis Biochemicals. Rabbit polyclonal anti actin antibody was obtained from Sigma Chemicals. Wild-type, chk2 inhibitor constitutively energetic and kinase dead individual GSK3B in pCMV Tag 5A expression vector were generously provided. Cell Lines and Cell Culture The human NSCLC cell lines found in this study were provided by Dr. R. Lotan in 2003 and cultured as previously described. H157 and A549 cell lines were recently authenticated by Genetica DNA Laboratories, Inc. by evaluation of the STR DNA profile. One other cell lines used haven’t been authenticated. The steady H157 Lac Z 5, H157 FLIPL 21 and H157 FLIPS 1 transfectants were described previously. Through the entire research, the concentrations of DMSO did not exceed 0. 05%. Western Blot Analysis Whole cell protein lysates were prepared and analyzed by Western blotting as described previously. Cell Survival Assay Cells were seeded in 96 well cell culture dishes and treated 24 hours later with all the agents mentioned. The viable cell number was determined as previously described, using the sulforhodamine W analysis.