Myeloid zinc finger 1 mediates sulindac sulfide-induced upregulation of death receptor 5 of human colon cancer cells

Abstract

A combined therapy of sulindac sulfide and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising strategy for the treatment of cancer. Sulindac sulfide had been shown to induce the expression of death receptor 5 (DR5), a receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism underlying the upregulation of DR5 has not yet been elucidated. We demonstrate here that myeloid zinc finger 1 (MZF1) mediates the induction of DR5 by sulindac sulfide. Sulindac sulfide induced the expression of DR5 at the protein and mRNA levels in colon cancer SW480 cells. Furthermore, sulindac sulfide increased DR5 promoter activity. We showed that sulindac sulfide stimulated DR5 promoter activity via the -301 to -253 region. This region contained a putative MZF1-binding site. Site-directed mutations in the site abrogated the enhancement in DR5 promoter activity by sulindac sulfide. MZF1 directly bound to the putative MZF1-binding site of the DR5 promoter and the binding was increased by sulindac sulfide. The expression of MZF1 was also increased by sulindac sulfide, and MZF1 siRNA attenuated the upregulation of DR5 by sulindac sulfide. These results indicate that sulindac sulfide induces the expression of DR5 by up-regulating MZF1.

Figure 1. Sulindac sulfide enhanced TRAIL-induced apoptosis in SW480 cells.

(a) SW480 cells were treated with 200 μM sulindac sulfide and/or the indicated concentrations of TRAIL for 24 h. Cells were analyzed for DNA content by PI staining (FL2-H) using a flow cytometer. The percentages of sub-G1 are shown as a bar graph. (b) DAPI staining of SW480 cells. SW480 cells were treated with 200 μM sulindac sulfide and/or 10 ng/ml TRAIL for 24 h. Nuclear morphology was visualized using DAPI staining under a fluorescence microscope. (c) SW480 cells were treated with 200 μM sulindac sulfide and/or 10 ng/ml TRAIL with or without 20 μM zVAD-fmk for 24 h. The effects were analyzed as described in (a). Data represent the means +/− S.D. of three determinations. *: p < 0.05 (d) Western blotting for caspase-8, caspase-3 or PARP. SW480 cells were treated with 200 μM sulindac sulfide and/or 10 ng/ml TRAIL for 24 h. β-actin was used as a loading control.

Figure 2. Sulindac sulfide induced DR5 expression in SW480 cells.

(a) RNase protection assay. SW480 cells were treated with or without 200 μM sulindac sulfide for 24 h. Total RNA from SW480 cells was hybridized with probes, and then digested with RNase as described in the Materials and Methods. The housekeeping genes GAPDH and ribosomal protein L32 are shown as controls. (b) Northern blot analysis. SW480 cells were treated with various concentrations of sulindac sulfide for 24 h. Total RNA was probed with human DR5 cDNA. Ethidium bromide staining of 28S and 18S rRNA are shown as loading controls. (c) Western blotting for DR5. SW480 cells were treated with the indicated concentrations of sulindac sulfide for 24 h. β-actin was used as a loading control. (d) Western blotting for DR5. Cells were treated with or without 200 μM sulindac sulfide for the period indicated. β-actin was used as a loading control. −, treated with solvent DMSO.

Figure 3. Sulindac sulfide induced DR5 promoter activity in SW480 cells.

SW480 cells were transiently transfected with reporter plasmids containing various sizes of DR5 promoters and the luciferase gene. Twenty-four hours after the transfection, cells were treated with or without 200 μM sulindac sulfide for 24 h, and cell lysates were the harvested for the luciferase assay, as described in the Materials and Methods. Relative luciferase activity is shown as raw light units (RLU) standardized with the protein concentrations. Data represent the means of triplicate experiments (bars, S.D.). −: treated with solvent DMSO. *: p < 0.05.

Figure 4. Analysis of sulindac sulfide-responsive elements in the DR5 promoter.

(a)(b) The luciferase assay was performed as described in Figure 3 with the indicated reporter plasmids. Data represent the means of triplicate experiments (bars, S.D.). +: treated with 200 μM sulindac sulfide for 24 h, −: treated with solvent DMSO. *: p < 0.05.

Figure 5. Mutation in the MZF1-binding site attenuated activation of the DR5 promoter due to sulindac sulfide.

(a) The sulindac sulfide-responsive region in the DR5 promoter and MZF1-binding sequences are shown. The predicted transcription factor-binding sites are shown using TFSEARCH 1.3. Mutation sequences are described in small letters. (b) The luciferase assay was performed as described in Figure 3 with the indicated reporter plasmids. Data represent the means of triplicate experiments (bars, S.D.). +: treated with 200 μM sulindac sulfide for 24 h, −: treated with solvent DMSO. *: p < 0.05 (c) Nuclear extract from cells treated with sulindac sulfide or DMSO were analyzed by gel shift assay, as described in Materials and methods. Increased amounts of the unlabeled oligonucleotides (×2 or ×8) were used as competitors. WT: wild-type MZF1-binding site of DR5 promoter. MT: mutant MZF1-binding site of DR5 promoter.

Figure 6. Sulindac sulfide induced MZF1 expression, and MZF1 siRNA reduced the enhancement in DR5 expression by sulindac sulfide.

(a) Western blotting for MZF1. SW480 cells were treated with the indicated concentrations of sulindac sulfide for 24 h. β-actin was used as a loading control. (b) The luciferase assay was performed as described in Figure 5 with the indicated reporter plasmids. The MZF1 overexpression plasmid pcDNA3.1MZF1 was co-transfected with luciferase reporter plasmids as described in the Materials and Methods. The fold induction in promoter activity (pcDNA3.1MZF1 vs control vector pcDNA3.1) was shown as a bar graph. Data represent the means +/− S.D. of three determinations. *: p < 0.05 (c) Western blotting for DR5 and MZF1. SW480 cells were transfected with MZF1 siRNA or negative control siRNA at 80 nM. Twenty-four hours after the transfection, cells were treated with or without 200 μM sulindac sulfide for 24 h. β-actin was used as a loading control. −: treated with DMSO, mock: transfection was performed without any siRNA.

Figure 7. The scheme of mechanisms enhancing DR5 expression by sulindac sulfide.

Sulindac sulfide increases MZF1. The transcription factor MZF1 then upregulates DR5 promoter activity through its binding site.