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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 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.
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f3: 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.

Mentions: We next examined whether sulindac sulfide affected gene expression related to cell death using the RNase protection assay. As shown in Figure 2a, sulindac sulfide increased DR5, DR4, and TRAIL mRNA levels. Among these, the upregulation of DR5 by sulindac sulfide was the most prominent. Therefore, we confirmed that sulindac sulfide increased DR5 mRNA level in a dose-dependent manner by Northern blotting (Fig. 2b). Sulindac sulfide also increased DR5 protein expression in a dose- and time-dependent manner (Fig. 2c and d). Using luciferase reporter plasmids carrying the DR5 promoter, we examined the mechanism underlying how sulindac sulfide up-regulated the expression of DR5 (Fig. 3). Previous studies reported that the transcription factors p53 and NF-κB increased DR5 promoter activity through these consensus elements on intron 133343536. With or without p53- and NF-κB- binding sites, DR5 promoter activity was enhanced by the sulindac sulfide treatment. These results demonstrated that sulindac sulfide up-regulated DR5 expression at a transcriptional level and the responsive element against sulindac sulfide was on the upstream promoter region of the DR5 gene.


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

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

f3: 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.
Mentions: We next examined whether sulindac sulfide affected gene expression related to cell death using the RNase protection assay. As shown in Figure 2a, sulindac sulfide increased DR5, DR4, and TRAIL mRNA levels. Among these, the upregulation of DR5 by sulindac sulfide was the most prominent. Therefore, we confirmed that sulindac sulfide increased DR5 mRNA level in a dose-dependent manner by Northern blotting (Fig. 2b). Sulindac sulfide also increased DR5 protein expression in a dose- and time-dependent manner (Fig. 2c and d). Using luciferase reporter plasmids carrying the DR5 promoter, we examined the mechanism underlying how sulindac sulfide up-regulated the expression of DR5 (Fig. 3). Previous studies reported that the transcription factors p53 and NF-κB increased DR5 promoter activity through these consensus elements on intron 133343536. With or without p53- and NF-κB- binding sites, DR5 promoter activity was enhanced by the sulindac sulfide treatment. These results demonstrated that sulindac sulfide up-regulated DR5 expression at a transcriptional level and the responsive element against sulindac sulfide was on the upstream promoter region of the DR5 gene.

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