J Breast Cancer.  2018 Mar;21(1):21-27. 10.4048/jbc.2018.21.1.21.

Berberine Suppresses Fibronectin Expression through Inhibition of c-Jun Phosphorylation in Breast Cancer Cells

• Jeong Y
• You D
• Kang HG
• Yu J
• Kim SW
• Nam SJ
• Lee JE
• Kim S

• KMID: 2441864
• DOI: http://doi.org/10.4048/jbc.2018.21.1.21

Abstract

PURPOSE
The exact mechanism regulating fibronectin (FN) expression in breast cancer cells has not been fully elucidated. In this study, we investigated the pharmacological mechanism of berberine (BBR) with respect to FN expression in triple-negative breast cancer (TNBC) cells.
METHODS
The clinical significance of FN mRNA expression was analyzed using the Kaplan-Meier plotter database (http://kmplot.com/breast). FN mRNA and protein expression levels were analyzed by real-time polymerase chain reaction and western blotting, respectively.
RESULTS
Using publicly available clinical data, we observed that high FN expression was associated with poor prognosis in patients with breast cancer. FN mRNA and protein expression was increased in TNBC cells compared with non-TNBC cells. As expected, recombinant human FN significantly induced cell spreading and adhesion in MDA-MB231 TNBC cells. We also investigated the regulatory mechanism underlying FN expression. Basal levels of FN mRNA and protein expression were downregulated by a specific activator protein-1 (AP-1) inhibitor, SR11302. Interestingly, FN expression in TNBC cells was dose-dependently decreased by BBR treatment. The level of c-Jun phosphorylation was also decreased by BBR treatment.
CONCLUSION
Our findings demonstrate that FN expression is regulated via an AP-1-dependent mechanism, and that BBR suppresses FN expression in TNBC cells through inhibition of AP-1 activity.

Keyword

Berberine; Cell adhesion; Fibronectins; Transcription factor AP-1

MeSH Terms

Berberine*
Blotting, Western
Breast Neoplasms*
Breast*
Cell Adhesion
Fibronectins*
Humans
Phosphorylation*
Prognosis
Real-Time Polymerase Chain Reaction
RNA, Messenger
Transcription Factor AP-1
Triple Negative Breast Neoplasms
Berberine
Fibronectins
RNA, Messenger
Transcription Factor AP-1

Figure

• 

  Figure 1 Aberrant fibronectin (FN) expression is associated with poor prognosis in breast cancer patients. Clinical relevance of FN mRNA expression obtained from a public database (Kaplan-Meier plotter database; http://kmplot.com/breast). (A) Relapse-free survival. (B) Overall survival. HR=hazard ratio.

• 

  Figure 2 Level of fibronectin (FN) expression and role of FN in breast cancer cells. After serum starvation for 24 hours, the levels of FN mRNA and protein expression were analyzed by real-time polymerase chain reaction (A) and western blotting (B), respectively. (C) MDA-MB231 cells were seeded with or without 100 ng/mL recombinant human FN for 3 hours. Cell morphology was analyzed using a CK40 inverted microscope. (D) Rates of cell adhesion were analyzed by adhesion assay, as described in Methods. The results are representative of three independent experiments. Values shown are mean±standard error of the mean. TNBC=triple-negative breast cancer. *p<0.01 vs. vehicle.

• 

  Figure 3 Basal fibronectin (FN) expression is suppressed by SR11302 treatment. (A) After serum starvation for 24 hours, cells were treated with 10 µM SR11302 for 24 hours. The level of FN mRNA expression was analyzed by real-time polymerase chain reaction. (B, C) After serum starvation for 24 hours, cells were treated with the indicated concentrations of SR11302 for 24 hours. The level of FN protein expression was analyzed by western blotting using conditioned culture media and lysates for both MDA-MB231 (B) and Hs578T (C) cells. The results are representative of three independent experiments. Values shown are mean±standard error of the mean. Con=control; SR=SR11302. *p<0.01 vs. con; †p<0.05.

• 

  Figure 4 Basal fibronectin (FN) expression is suppressed by berberine (BBR) treatment. (A) The chemical structure of BBR. (B) After serum starvation for 24 hours, MDA-MB231 and Hs578T cells were treated with 50 µM BBR for 24 hours, and the level of FN mRNA expression was analyzed by realtime polymerase chain reaction. (C, D) After serum starvation for 24 hours, cells were treated with the indicated concentrations of BBR for 24 hours. The level of FN protein expression was analyzed by western blotting using conditioned culture media and lysates of MDA-MB231 (C) and Hs578T (D) cells. (E) After serum starvation for 24 hours, cells were treated with 50 µM BBR for 4 hours. The levels of p-c-Jun, c-Fos, and β-actin expression in Hs578T cells were analyzed by western blotting. The results are representative of three independent experiments. Values shown are mean±standard error of the mean. Con=control. *p<0.01 vs. con; †p<0.05.

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