Fig 2: Effect of NSAID on expression of CHOP/DR5 and c-FLIP and autophagic degradation of c-FLIP in HCC cells. (A) SNU-475/TR cells were treated with serial doses of CCB or DMC for 24 h. (B) SNU-475/TR cells were pretreated with 5 µM chloroquine (CQ), 10 mM 3-methyladenine (3-MA) or 5 µM LY294002 (LY) for 3 h followed by treatment of 40 µM CCB for 24 h. (C) SNU-354 cells were treated with serial doses of CCB or DMC for 24 h or 5 µM LY for 3 h followed with treatment of 40 µM CCB for 24 h. Subsequently, altered levels of the indicted molecules in these cells were determined by western blot analysis. (D) SNU-475/TR and SNU-449 cells were treated with or without 50 µM CCB for 24 h, and were collected at 0, 3 and 6 h following treatment with 20 µg/ml cycloheximide (CHX) and the level of c-FLIPL was determined by western blot analysis. TRAIL, TNF-related apoptosis inducing ligand; NSAID, non-steroidal anti-inflammatory drug; CHOP, C/EBP homologous protein; DR5, death receptor TRAIL-R2; c-FLIP, cellular-FLICE inhibitory protein; HCC, hepatocellular carcinoma; CCB, celecoxib; DMC, 2,5-dimethyl celecoxib.

Fig 3: Comparison of cytotoxicity, activity of caspases and surface expression of DR5, CD44 and CD133 in SNU-475/TR cells and other HCC cells in the absence and presence of TRAIL. (A) SNU-475/TR cells and the parental SNU-475 cells were treated with various concentrations of TRAIL (0. 1. 10. 100 and 200 ng/ml). Percentage of cell survival was determined after 96 h of incubation using MTT assay. Each bar represents the mean ± SD of triplicate experiments. **P<0.01, ***P<0.001 compared to the SNU-475 cells. (B) SNU-475/TR and SNU-475 cells were treated with TRAIL (1 or 10 ng/ml) for 24 h, and the level of activation by the cleavage (Cl.) of pro-caspase-8 (Casp8), -3 (Casp3), -9 (Casp9) and PARP and the expression of CD44/CD133 were determined by western blot analysis. Tubulin was used as a loading control. (C) Both cell lines were stained with anti-DR5 antibody to determine the surface expression of DR5 by a flow cytometer. (D) Four HCC and SNU-475/TR cell lines were stained with anti-CD133 or anti-CD44 antibody to determine the surface expression of CD44 or CD133 by a flow cytometer. HCC, hepatocellular carcinoma; TRAIL, TNF-related apoptosis inducing ligand; DR4, death receptor TRAIL-R1; PARP, poly(ADP-ribose) polymerase.

Fig 4: CCB-induced regulation of DR5/c-FLIP and CSC markers through the ATF/CHOP signaling axis in SNU-475/TR cells, and the effect of NSAID on the cell surface expression of DR5 in HCC cells. (A) The altered levels of ATF4, CHOP, DR5 (mature form), c-FLIPL/S, CD44, ALDH1 and Nanog in SNU-475 and SNU-475/TR cells by CCB treatment (40 µM for 24 h) were determined by western blot analysis. Actin was used as a loading control. (B-D) HCC cell lines were treated with the indicated dose of CCB or DMC for 24 h. The cells were stained with control IgG or anti-DR5 antibody and subsequently labeled with PE-conjugated secondary antibodies to determine the surface expression of DR5, and the cell surface expression were measured by a flow cytometer. CCB, celecoxib; DR5, death receptor TRAIL-R2; c-FLIP, cellular-FLICE inhibitory protein; CSC, cancer stem cell; ATF, activating transcription factor; CHOP, C/EBP homologous protein; ALDH1, aldehyde dehydrogenase 1; NSAID, non-steroidal anti-inflammatory drug; HCC, hepatocellular carcinoma; DMC, 2,5-dimethyl celecoxib.