ROS and CHOP are critical for dibenzylideneacetone to sensitize tumor cells to TRAIL through induction of death receptors and downregulation of cell survival proteins

Abstract

Because tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively kills tumor cells, it is being tested in cancer patients. Unfortunately, patients develop resistance to the cytokine, therefore, agents that can sensitize cells to TRAIL are urgently needed. In this study, we investigated whether dibenzylideneacetone (DBA) can sensitize cancer cells to TRAIL and potentiates TRAIL-induced apoptosis. As indicated by accumulation of the membrane phospholipid phosphatidylserine, DNA breaks, intracellular esterase activity, and activation of caspase-8, -9, and -3, we concluded that DBA potentiated TRAIL-induced apoptosis in colon cancer cells. DBA also converted TRAIL resistant-cells to TRAIL-sensitive. When examined for the mechanism, we found that DBA decreased the expression of antiapoptotic proteins and decoy receptor-2 and increased proapoptotic proteins. DBA also induced both death receptor (DR)-5 and DR4. Knockdown of DR5 and DR4 by small interfering RNA (SiRNA) reduced the sensitizing effect of DBA on TRAIL-induced apoptosis. In addition, DBA increased the expression of CHOP proteins. Knockdown of CHOP by siRNA decreased the induction of DBA-induced DR5 expression and apoptosis. Induction of receptors by DBA, however, was p53-independent, as deletion of p53 had no effect on receptor induction. We observed that DBA-induced induction of DR5 and DR4 was mediated through generation of reactive oxygen species (ROS), as N-acetylcysteine blocked the induction of death receptors and suppression of cell survival proteins by DBA. Overall, our results show that DBA potentiates TRAIL-induced apoptosis through downregulation of cell survival proteins and upregulation of death receptors via activation of ROS and CHOP mediated pathways.

Figure 1

DBA-potentiated TRAIL induced apoptosis of HCT116 cells. (A) Chemical structure of DBA. (B) Cells were pretreated with 15 µM of DBA for 12 h the media were removed, and the cells then exposed TRAIL for 24 h. Cell viability was then analyzed by the MTT method (Left panel) and cell death by the Live/Dead assay (Right panel) using indicated concentration of DBA. Green is live and red is dead cells. Percent dead cells are mentioned below the photo. * indicates significant over control at P<0.001. (C) Cells were treated with 15 µM of DBA and TRAIL as described above. Cells were stained with PI/Annexin V (Upper panel) and PI alone (Lower panel) and then analyzed by FACS. (D) Whole-cell extracts were prepared and analyzed by Western blotting using antibodies against caspase-8, caspase-3, caspase-9 and PARP.

Figure 2

Effects of DBA on antiapoptotic, pro-apoptotic and GSK-3β expression. HCT116 cells were pretreated with indicated dose of DBA for 24 h. Whole-cell extracts were prepared and analyzed by Western blotting using the antibodies against antiapoptotic (A), pro-apoptotic (B) proteins, and GSK-3β (C). The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading.

Figure 3

DBA induces expression of death receptors and decoy receptors. (A) HCT116 cells (1×10⁶ cells/well) were treated with indicated dose (Left panel) of DBA and time (Right panel). Whole-cell extracts were then prepared and analyzed by Western blotting. (B) HCT116 cells were treated with 15 µM DBA for 24 h and analyzed for cell surface DR4 and DR5 by immunofluorescent staining and subsequent flow cytometry. Filled yellow peaks, cells stained with a matched control phycoerythrin-conjugated IgG isotype antibody. (B) HCT116 cells were pretreated with indicated dose of DBA for 24 h. Whole-cell extracts were prepared and subjected for Western blotting. (D) DBA upregulated DR5 and DR4 in various types of cancer cells. Cells (1×10⁶ cells) were treated with 15 µM DBA for 24 h, after which whole-cell extracts were prepared and analyzed by Western blotting. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading.

Figure 4

Involvement of DRs on DBA-induced sensitization of TRAIL. (A) HCT116 cells were transfected with DR5 siRNA, DR4 siRNA alone or combined and control siRNA. After 48 h, cells were treated with 15 µM DBA for 24 h, and whole- cell extracts were prepared for Western blotting for DR5 and DR4. (B) Cells were seeded in a chamber slide and transfected with siRNAs. After 48 h, cells were pretreated with 15 µM of DBA for 12 h the media were removed, and then exposed to TRAIL (25 ng/mL) for 24 h. Cell death was determined by the Live/Dead Assay. Green is live and red is dead cells. Percent dead cells are mentioned below the photo.

Figure 5

Upregulation of death receptors are p53, ERK1/2 and JNK independent. (A) HCT116 (p53 parental and p53 knockout) cells (1 × 10⁶/well) were treated with 15 µM DBA for 24 h. Whole-cell extracts were prepared and analyzed by Western blotting using p53 and DR5 antibodies (Left panel) and p53 antibody (Right panel). The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. (B) HCT116 cells were treated as indicated above and subjected to Western blotting for phosphorylated Akt1/2, ERK1/2 and JNK (Left panel) and PPARγ (Right panel), (C) CHOP. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. (D) HCT116 cells were transfected with CHOP siRNA and control siRNA. After 48 h, cells were treated with 15 µM DBA for 24 h, and whole- cell extracts were prepared for Western blotting (Left panel). Cells were seeded in a chamber slide and transfected with CHOP siRNAs and treated with DBA and TRAIL as indicated above. Cell death was determined by the Live/Dead Assay (Right panel). Green is live and red is dead cells. Percent dead cells are mentioned below the photo.

Figure 6

DBA induces generation of ROS and DBA-induced up-regulation of DR5 and DR4 was mediated by ROS. (A) HCT116 (1×10⁶ cells) cells were labeled with DCF-DA, treated with indicated concentration of DBA for 1 h and examined for ROS production by flow cytometer (Left panel). HCT116 cells (1×10⁶ cells) were pretreated with various concentrations of NAC for 1 h and then the cells were treated with 15 µM DBA for 24 h. Whole-cell extracts were prepared and analyzed by Western blotting (Right panel). (B) NAC reverses the DBA-induced inhibition of antiapoptotic proteins. HCT116 cells were pretreated with NAC for 1 h and then treated with 15 µM DBA for 24 h. Whole-cell extracts were prepared and subjected to Western blotting. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. (C) NAC reverses cell death induced by combination of DBA and TRAIL. HCT116 cells were pretreated with NAC (10 mM) for 1 h and then treated with 15 µM DBA for 12 h. After washing with PBS cells were treated with TRAIL (25 ng/mL) for 24 h. Cell death was determined by the Live/Dead assay. Percent dead cells are mentioned below the photo. (D) NAC inhibited caspase activation and PARP cleavage induced by combination of TRAIL and DBA. HCT116 cells were treated with NAC, DBA and TRAIL as indicated above. Whole-cell extracts were prepared and analyzed by Western blotting using the relevant antibodies. β-actin was used as a loading control.

Figure 7

DBA sensitizes TRAIL resistance cells and induces apoptosis. HT29 cells were pretreated with of DBA (15 µM) for 12 h. After removal of the media cells were exposed to TRAIL for 24 h. (A) Cell death was analyzed by the Live/Dead assay. Green is live and red is dead cells. Percent dead cells are mentioned below the photo. * indicates significant over control at P<0.001. (B) Cells were stained with PI for FACS analysis (Left panel) and cell viability was determined by MTT assay (Right panel). (C) Whole cell extract were prepared and subjected for western blotting using relevant antibodies.