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

Horinaka M, Yoshida T, Tomosugi M, Yasuda S, Sowa Y, Sakai T - Sci Rep (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan [2].

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.

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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.
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f6: 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.

Mentions: As described above, MZF1 was suggested to contribute to the enhancement of the DR5 promoter by sulindac sulfide. We investigated the behavior of MZF1 when treated with sulindac sulfide. The expression of MZF1 was increased by the sulindac sulfide treatment (Fig. 6a). Thus, we examined whether MZF1 induction was related to the expression of DR5. In another human colon cancer HCT116 cells, the expressions of DR5 and MZF1 were similarly induced by sulindac sulfide (Supplemental Figure). The overexpression of MZF1 increased DR5 promoter activity, whereas the DR5 promoter with a mutation in the MZF1-binding site was not activated by the overexpression of MZF1 (Fig. 6b). We also examined the MZF1 knockdown effect on the upregulation of DR5 by sulindac sulfide. MZF1 siRNA reduced the upregulation of DR5 by sulindac sulfide (Fig. 6c), which indicated that MZF1 at least partially mediated the upregulation of DR5 induced by sulindac sulfide.


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

Horinaka M, Yoshida T, Tomosugi M, Yasuda S, Sowa Y, Sakai T - Sci Rep (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4126006&req=5

f6: 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.
Mentions: As described above, MZF1 was suggested to contribute to the enhancement of the DR5 promoter by sulindac sulfide. We investigated the behavior of MZF1 when treated with sulindac sulfide. The expression of MZF1 was increased by the sulindac sulfide treatment (Fig. 6a). Thus, we examined whether MZF1 induction was related to the expression of DR5. In another human colon cancer HCT116 cells, the expressions of DR5 and MZF1 were similarly induced by sulindac sulfide (Supplemental Figure). The overexpression of MZF1 increased DR5 promoter activity, whereas the DR5 promoter with a mutation in the MZF1-binding site was not activated by the overexpression of MZF1 (Fig. 6b). We also examined the MZF1 knockdown effect on the upregulation of DR5 by sulindac sulfide. MZF1 siRNA reduced the upregulation of DR5 by sulindac sulfide (Fig. 6c), which indicated that MZF1 at least partially mediated the upregulation of DR5 induced by sulindac sulfide.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan [2].

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.

Show MeSH
Related in: MedlinePlus