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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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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.
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f5: 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.

Mentions: We examined the predicted transcription factor-binding sites using TFSEARCH. This sequence represents the possible binding region for the transcription factor MZF1 (Fig. 5a). We introduced a site-directed mutation to the putative MZF1-binding site on the DR5 promoter and generated pDR5/mtMZF1 (Fig. 5a). As shown in Figure 5b, sulindac sulfide did not enhance the promoter activity of pDR5/mtMZF1.


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)

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

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

f5: 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.
Mentions: We examined the predicted transcription factor-binding sites using TFSEARCH. This sequence represents the possible binding region for the transcription factor MZF1 (Fig. 5a). We introduced a site-directed mutation to the putative MZF1-binding site on the DR5 promoter and generated pDR5/mtMZF1 (Fig. 5a). As shown in Figure 5b, sulindac sulfide did not enhance the promoter activity of pDR5/mtMZF1.

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