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Glaucine inhibits breast cancer cell migration and invasion by inhibiting MMP-9 gene expression through the suppression of NF-κB activation.

Kang H, Jang SW, Pak JH, Shim S - Mol. Cell. Biochem. (2015)

Bottom Line: We further show that glaucine significantly blocks phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression and activity in a dose-dependent manner.Moreover, glaucine attenuates PMA-induced IκBα degradation and nuclear translocation of NF-κB.Taken together, our findings indicate that the MMP-9 inhibitory activity of glaucine and its abilities to attenuate IκBα and NF-κB activities may be therapeutically useful as a novel means of controlling breast cancer growth and invasiveness.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul, 138-736, Republic of Korea.

ABSTRACT
Matrix metalloproteinase-9 (MMP-9) plays a central role in the invasion and metastasis of various types of cancer cells. Here, we demonstrate that glaucine, an alkaloid isolated from the plant Corydalis turtschaninovii tuber (Papaveraceae), can inhibit the migration and invasion of human breast cancer cells. We further show that glaucine significantly blocks phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression and activity in a dose-dependent manner. Results from reporter gene and electrophoretic mobility shift assays revealed that glaucine inhibits MMP-9 expression by suppressing activation of the nuclear transcription factor nuclear factor-κB (NF-κB). Moreover, glaucine attenuates PMA-induced IκBα degradation and nuclear translocation of NF-κB. Finally, we also found that glaucine inhibits invasion and MMP-9 expression in the highly metastatic MDA-MB-231 breast cancer cell line. Taken together, our findings indicate that the MMP-9 inhibitory activity of glaucine and its abilities to attenuate IκBα and NF-κB activities may be therapeutically useful as a novel means of controlling breast cancer growth and invasiveness.

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Glaucine inhibits the transcriptional activity of MMP-9 promoter via suppression of PMA-stimulated NF-κB activity. a MCF-7 cells were transfected with the pMMP-9-luciferase and the pSV40-β-galactosidase vectors. The transfected cells were treated with the indicated concentrations of glaucine for 30 min and stimulated with 100 nM PMA for 9 h. The luciferase activity was normalized by β-galactosidase activity. Each value represents the mean ± SD of three independent experiments and is expressed relative to a control. b MCF-7 cells were transfected with NF-κB binding site mutant (mNF-κB) MMP-9-Luc. The transfected cells were treated with glaucine for 30 min and stimulated with 100 nM PMA for 9 h. The luciferase activity was normalized by β-galactosidase activity. Each value represents the mean ± SD of three independent experiments and is expressed relative to a control. One-way ANOVA was performed to determine statistical significance (*P < 0.05). c MCF-7 cells were transfected with the reporter plasmids containing tandem NF-κB binding sites. After 24 h, cells were treated with or without 100 nM PMA for 9 h and the luciferase activities were determined. Each value represents the mean ± SD of three independent experiments and is expressed relative to a control. One-way ANOVA was performed to determine statistical significance (*P < 0.05)
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Fig4: Glaucine inhibits the transcriptional activity of MMP-9 promoter via suppression of PMA-stimulated NF-κB activity. a MCF-7 cells were transfected with the pMMP-9-luciferase and the pSV40-β-galactosidase vectors. The transfected cells were treated with the indicated concentrations of glaucine for 30 min and stimulated with 100 nM PMA for 9 h. The luciferase activity was normalized by β-galactosidase activity. Each value represents the mean ± SD of three independent experiments and is expressed relative to a control. b MCF-7 cells were transfected with NF-κB binding site mutant (mNF-κB) MMP-9-Luc. The transfected cells were treated with glaucine for 30 min and stimulated with 100 nM PMA for 9 h. The luciferase activity was normalized by β-galactosidase activity. Each value represents the mean ± SD of three independent experiments and is expressed relative to a control. One-way ANOVA was performed to determine statistical significance (*P < 0.05). c MCF-7 cells were transfected with the reporter plasmids containing tandem NF-κB binding sites. After 24 h, cells were treated with or without 100 nM PMA for 9 h and the luciferase activities were determined. Each value represents the mean ± SD of three independent experiments and is expressed relative to a control. One-way ANOVA was performed to determine statistical significance (*P < 0.05)

Mentions: NF-κB plays an important role in regulating MMP-9 expression in various cancer cell lines [19]. We examined the effects of glaucine on the binding of this transcription factor to the MMP-9 promoter. A luciferase reporter gene containing the MMP-9 promoter region (−925/+13) was transiently transfected into MCF-7 cells, and luciferase activity was determined. MMP-9 promoter activity in cells treated with PMA was upregulated by 3.3-fold compared with that in untreated cells. Glaucine inhibited PMA-induced MMP-9 promoter activity in a dose-dependent manner (Fig. 4a). The transcription factor binding sites in the MMP-9 promoter include sites for NF-κB (−600 bp) [20]. To determine whether NF-κB is involved in MMP-9 transcription regulation, we generated a MMP-9 promoter with a mutation in the NF-κB (−600 bp) binding site. The results of the reporter gene assay showed that PMA-induced promoter activity of the mutated promoter in the NF-κB biding site (-925/+ 13) was not affected by glaucine (Fig. 4b). We also performed experiments using luciferase reporter vectors containing tandem repeats of the NF-κB binding sites and observed dose-dependent reductions in luciferase activity in cells transfected with the NF-κB reporter, following glaucine treatment (Fig. 4c). These results indicated that NF-κB binding to the MMP-9 promoter region contributed to the inhibitory effect of glaucine on PMA-induced MMP-9 transcription.Fig. 4


Glaucine inhibits breast cancer cell migration and invasion by inhibiting MMP-9 gene expression through the suppression of NF-κB activation.

Kang H, Jang SW, Pak JH, Shim S - Mol. Cell. Biochem. (2015)

Glaucine inhibits the transcriptional activity of MMP-9 promoter via suppression of PMA-stimulated NF-κB activity. a MCF-7 cells were transfected with the pMMP-9-luciferase and the pSV40-β-galactosidase vectors. The transfected cells were treated with the indicated concentrations of glaucine for 30 min and stimulated with 100 nM PMA for 9 h. The luciferase activity was normalized by β-galactosidase activity. Each value represents the mean ± SD of three independent experiments and is expressed relative to a control. b MCF-7 cells were transfected with NF-κB binding site mutant (mNF-κB) MMP-9-Luc. The transfected cells were treated with glaucine for 30 min and stimulated with 100 nM PMA for 9 h. The luciferase activity was normalized by β-galactosidase activity. Each value represents the mean ± SD of three independent experiments and is expressed relative to a control. One-way ANOVA was performed to determine statistical significance (*P < 0.05). c MCF-7 cells were transfected with the reporter plasmids containing tandem NF-κB binding sites. After 24 h, cells were treated with or without 100 nM PMA for 9 h and the luciferase activities were determined. Each value represents the mean ± SD of three independent experiments and is expressed relative to a control. One-way ANOVA was performed to determine statistical significance (*P < 0.05)
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Related In: Results  -  Collection

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Fig4: Glaucine inhibits the transcriptional activity of MMP-9 promoter via suppression of PMA-stimulated NF-κB activity. a MCF-7 cells were transfected with the pMMP-9-luciferase and the pSV40-β-galactosidase vectors. The transfected cells were treated with the indicated concentrations of glaucine for 30 min and stimulated with 100 nM PMA for 9 h. The luciferase activity was normalized by β-galactosidase activity. Each value represents the mean ± SD of three independent experiments and is expressed relative to a control. b MCF-7 cells were transfected with NF-κB binding site mutant (mNF-κB) MMP-9-Luc. The transfected cells were treated with glaucine for 30 min and stimulated with 100 nM PMA for 9 h. The luciferase activity was normalized by β-galactosidase activity. Each value represents the mean ± SD of three independent experiments and is expressed relative to a control. One-way ANOVA was performed to determine statistical significance (*P < 0.05). c MCF-7 cells were transfected with the reporter plasmids containing tandem NF-κB binding sites. After 24 h, cells were treated with or without 100 nM PMA for 9 h and the luciferase activities were determined. Each value represents the mean ± SD of three independent experiments and is expressed relative to a control. One-way ANOVA was performed to determine statistical significance (*P < 0.05)
Mentions: NF-κB plays an important role in regulating MMP-9 expression in various cancer cell lines [19]. We examined the effects of glaucine on the binding of this transcription factor to the MMP-9 promoter. A luciferase reporter gene containing the MMP-9 promoter region (−925/+13) was transiently transfected into MCF-7 cells, and luciferase activity was determined. MMP-9 promoter activity in cells treated with PMA was upregulated by 3.3-fold compared with that in untreated cells. Glaucine inhibited PMA-induced MMP-9 promoter activity in a dose-dependent manner (Fig. 4a). The transcription factor binding sites in the MMP-9 promoter include sites for NF-κB (−600 bp) [20]. To determine whether NF-κB is involved in MMP-9 transcription regulation, we generated a MMP-9 promoter with a mutation in the NF-κB (−600 bp) binding site. The results of the reporter gene assay showed that PMA-induced promoter activity of the mutated promoter in the NF-κB biding site (-925/+ 13) was not affected by glaucine (Fig. 4b). We also performed experiments using luciferase reporter vectors containing tandem repeats of the NF-κB binding sites and observed dose-dependent reductions in luciferase activity in cells transfected with the NF-κB reporter, following glaucine treatment (Fig. 4c). These results indicated that NF-κB binding to the MMP-9 promoter region contributed to the inhibitory effect of glaucine on PMA-induced MMP-9 transcription.Fig. 4

Bottom Line: We further show that glaucine significantly blocks phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression and activity in a dose-dependent manner.Moreover, glaucine attenuates PMA-induced IκBα degradation and nuclear translocation of NF-κB.Taken together, our findings indicate that the MMP-9 inhibitory activity of glaucine and its abilities to attenuate IκBα and NF-κB activities may be therapeutically useful as a novel means of controlling breast cancer growth and invasiveness.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul, 138-736, Republic of Korea.

ABSTRACT
Matrix metalloproteinase-9 (MMP-9) plays a central role in the invasion and metastasis of various types of cancer cells. Here, we demonstrate that glaucine, an alkaloid isolated from the plant Corydalis turtschaninovii tuber (Papaveraceae), can inhibit the migration and invasion of human breast cancer cells. We further show that glaucine significantly blocks phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression and activity in a dose-dependent manner. Results from reporter gene and electrophoretic mobility shift assays revealed that glaucine inhibits MMP-9 expression by suppressing activation of the nuclear transcription factor nuclear factor-κB (NF-κB). Moreover, glaucine attenuates PMA-induced IκBα degradation and nuclear translocation of NF-κB. Finally, we also found that glaucine inhibits invasion and MMP-9 expression in the highly metastatic MDA-MB-231 breast cancer cell line. Taken together, our findings indicate that the MMP-9 inhibitory activity of glaucine and its abilities to attenuate IκBα and NF-κB activities may be therapeutically useful as a novel means of controlling breast cancer growth and invasiveness.

Show MeSH
Related in: MedlinePlus