Downregulation of DcR3 sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis

Figures & data

Figure 1 Expression of DcR3 in hepatocellular carcinoma cell lines and sensitivity to TRAIL for HepG2 and BEL-7402.

Notes: (A) The expression of DcR3 in different hepatocellular carcinoma cells was quantified by qPCR and measured by ELISA. The differences among cell lines were significant (P<0.01). (B) The sensitivity of HepG2 and BEL-7402 cells to TRAIL (100 ng/mL) was measured by Annexin V. TRAIL had no significant effect on HepG2 (P>0.05), but the apoptosis of BEL-7402 increased. DMSO was used as control. Data in the bar graphs represent the mean ± SD of three repeated experiments.
Abbreviations: DcR, decoy receptor; ELISA, enzyme-linked immunosorbent assay; qPCR, quantitative polymerase chain reaction; SD, standard deviation; TRAIL, tumor-necrosis factor-related apoptosis-inducing ligand; DMSO, dimethyl sulfoxide.

Figure 2 Expression of DcR3 and apoptosis in hepatocellular carcinoma cell lines with different treatments.

Notes: (A) The expression of DcR3 in LV-RNAi and LV-NC cell lines was measured by qPCR or ELISA. The differences among cell lines were significant (P<0.001). (B) The apoptosis of four different cells (HepG2-NC, HepG2-SiDcR3, BEL-7402-NC, and BEL-7402-SiDcR3) treated with TRAIL (100 ng/mL) was measured by Annexin V. DMSO was used as control. Data in the bar graphs represent the mean ± SD of three repeated experiments.
Abbreviations: DcR, decoy receptor; ELISA, enzyme-linked immunosorbent assay; qPCR, quantitative polymerase chain reaction; SD, standard deviation; TRAIL, tumor-necrosis factor-related apoptosis-inducing ligand; DMSO, dimethyl sulfoxide.

Figure 3 Effect of TRAIL on cell viability and cell cycle.

Notes: (A) Effect of TRAIL on cell cycle of cells in various groups (BEL-7402 LV-NC + DMSO, BEL-7402 LV-NC + TRAIL, BEL-7402 LV-siRNA + DMSO, and BEL-7402 LV-siRNA + TRAIL). The differences among cell lines were significant (P<0.05). The highest percentage of LV-siRNA + TRAIL cells was arrested at G0/G1 phase. (B) Effect of TRAIL on cell viability of cells in various groups using CCK-8. The differences among cell lines were significant (P<0.05). All detections were repeated three times and the mean values were used for comparison.
Abbreviations: CCK8, cell counting kit 8; CV, variable coefficient; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; DMSO, dimethyl sulfoxide.

Figure 4 SiDcR3-combined TRAIL activated extrinsic and intrinsic apoptotic signaling pathways.

Notes: (A–F): SiDcR3-combined TRAIL activated extrinsic and intrinsic apoptotic signaling pathways, inhibited caspase-8, and prevented DcR3-TRAIL-induced cascade activation. HepG2 cells were pretreated with 50 μM Ac-IETD-CHO or 50 μM Ac-LEHD-CMK 20 min before treatment with TRAIL (100 ng/mL, for 24 h). Caspase-3, caspase-8, and caspase-9 activity was determined fluorometrically in cell extracts using Ac-DEVD-AMC, Ac-IETD-AFC, and Ac-LEHD-AMC as flurogenic substrate, respectively.
Abbreviations: DcR, decoy receptor; h, hours; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

Figure 5 SiDcR3 sensitizes hepatocellular carcinoma cells through Bcl-2 family members.

Notes: (A) The prosurvival Bcl-2 family members Bcl-2, Bcl-xL, IAP-2, survivin, and Mcl-1 and proapoptosis member Bax were examined using Western blot analysis after treatment with TRAIL (100 ng/mL) for 24 h. GAPDH was used as loading control. (B–G) The ratio of each protein to GAPHD. The differences among different proteins were significant (P<0.05). Data in the bar graphs represent the mean ± SD of three repeated experiments.
Abbreviations: DcR, decoy receptor; h, hours; SD, standard deviation; CV, variable coefficient; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

Figure 6 Silencing DcR3 combined TRAIL (100 ng/mL) can increase the expression of DR5.

Notes: (A) The expression of DR4 and DR5 was examined by Western blot. GAPHD was used as a loading control. The differences of DR5 among groups were significant (P<0.001). (B) HepG2 cells pretreated with neutralizing antibodies against DR4 and DR5 for 1 h and treated with TRAIL (100 ng/mL) for 24 h. Apoptosis was analyzed by Annexin-V/PI assay. (C) The caspase-8 activity was determined fluorometrically in cell extracts using Ac-IETD-AFC as the fluorogenic substrate. P<0.01. Data in the bar graphs represent the mean ± SD of three repeated experiments.
Abbreviations: DcR, decoy receptor; h, hours; PI, propidium iodide; SD, standard deviation; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

Figure 7 DcR3 and TRAIL-induced apoptosis.

Abbreviations: DcR, decoy receptor; FADD, Fas-associated protein with death domain; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.