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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 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.
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f1: 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.

Mentions: We examined the effect of sulindac sulfide on TRAIL-induced apoptosis by measuring the sub-G1 population, which reflected hypodiploid cells. Sulindac sulfide or TRAIL alone slightly induced apoptosis in SW480 colon cancer cells; however, the combined treatment with sulindac sulfide and TRAIL markedly induced apoptosis (Fig. 1a). As a result of DAPI staining, the combined treatment with sulindac sulfide and TRAIL induced condensed nuclei (Fig. 1b). The combined effect was blocked by the pan-caspase inhibitor zVAD-fmk, which indicated that apoptosis was caspase-dependent (Fig. 1c). Moreover, the combination cleaved caspase-8, caspase-3 and PARP (Fig. 1d). These results indicate that sulindac sulfide enhanced the efficacy of TRAIL to induce apoptosis and overcame TRAIL resistance in SW480 colon cancer cells.


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

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

f1: 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.
Mentions: We examined the effect of sulindac sulfide on TRAIL-induced apoptosis by measuring the sub-G1 population, which reflected hypodiploid cells. Sulindac sulfide or TRAIL alone slightly induced apoptosis in SW480 colon cancer cells; however, the combined treatment with sulindac sulfide and TRAIL markedly induced apoptosis (Fig. 1a). As a result of DAPI staining, the combined treatment with sulindac sulfide and TRAIL induced condensed nuclei (Fig. 1b). The combined effect was blocked by the pan-caspase inhibitor zVAD-fmk, which indicated that apoptosis was caspase-dependent (Fig. 1c). Moreover, the combination cleaved caspase-8, caspase-3 and PARP (Fig. 1d). These results indicate that sulindac sulfide enhanced the efficacy of TRAIL to induce apoptosis and overcame TRAIL resistance in SW480 colon cancer cells.

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