J Korean Neurosurg Soc.  2016 Mar;59(2):106-116. 10.3340/jkns.2016.59.2.106.

Bacitracin Inhibits the Migration of U87-MG Glioma Cells via Interferences of the Integrin Outside-in Signaling Pathway

• Li S
• Li C
• Ryu HH
• Lim SH
• Jang WY
• Jung S

• KMID: 2192039
• DOI: http://doi.org/10.3340/jkns.2016.59.2.106

Abstract

OBJECTIVE
Protein disulfide isomerase (PDI) acts as a chaperone on the cell surface, and it has been reported that PDI is associated with the tumor cell migration and invasion. The aims of this study are to investigate the anti-migration effect of bacitracin, which is an inhibitor of PDI, and the associated factor in this process.
METHODS
U87-MG glioma cells were treated with bacitracin in 1.25, 2.5, 3.75, and 5.0 mM concentrations. Western blot with caspase-3 was applied to evaluate the cytotoxicity of bacitracin. Adhesion, morphology, migration assays, and organotypic brain-slice culture were performed to evaluate the effect of bacitracin to the tumor cell. Western blot, PCR, and gelatin zymography were performed to investigate the associated factors. Thirty glioma tissues were collected following immunohistochemistry and Western blot.
RESULTS
Bacitracin showed a cytotoxicity in 3rd (p<0.05) and 4th (p<0.001) days, in 5.0 Mm concentration. The cell adhesion significantly decreased and the cells became a round shape after treated with bacitracin. The migration ability, the expression of phosphorylated focal adhesion kinase (p-FAK) and matrix metalloproteinase-2 (MMP-2) decreased in a bacitracin dose- and time-dependent manner. The U87-MG cells exhibited low-invasiveness in the 2.5 mM, compared with the untreated in organotypic brain-slice culture. PDI was expressed in the tumor margin, and significantly increased with histological glioma grades (p<0.001).
CONCLUSION
Bacitracin, as a functional inhibitor of PDI, decreased the phosphorylated FAK and the secreted MMP-2, which are the downstream of integrin and play a major role in cell migration and invasion, might become one of the feasible therapeutic strategies for glioblastoma.

Keyword

Bacitracin; Protein disulfide isomerase; Integrin; Glioma; Migration; Invasion

MeSH Terms

Bacitracin*
Blotting, Western
Caspase 3
Cell Adhesion
Cell Movement
Focal Adhesion Protein-Tyrosine Kinases
Gelatin
Glioblastoma
Glioma*
Immunohistochemistry
Matrix Metalloproteinase 2
Polymerase Chain Reaction
Protein Disulfide-Isomerases
Bacitracin
Caspase 3
Focal Adhesion Protein-Tyrosine Kinases
Gelatin
Matrix Metalloproteinase 2
Protein Disulfide-Isomerases

Figure

• 

  Fig. 1 PDI is related to tumor cell motility and it is localized in the cell cytosol and on the cell surface. A : The cell migration distance is measured with microscopy every 2 hours. The chart (right) shows the mobility of each cell lines (100×magnification). The distance between two sides is measured at five sites for the indicated periods of time. U87-MG, U118, and U251 have high motility, compare with U343MG-A. B : The same cell lines are used in the invasion assay, the invaded cells are counted after 24 hours (200×magnification). The experiment is performed 3 times, each data point represents the mean+SD of 4 cell lines, at the right. C : The expression of total PDI on the cell surface is shown by Western blots, and the fold difference between these four cell lines is displayed in the lower portion of the chart. D : PDI expression is observed in monolayer culture of U87-MG cells by immunofluorescence. Images are obtained by confocal laser scanning microscopy (400×magnification). *p<0.05, ***p<0.001. PDI : protein disulfide isomerase.

• 

  Fig. 2 A high Bacitracin concentration induces tumor cell apoptosis. A and B : No difference in caspase-3 expression was observed on days 1 and 2 after bacitracin treatment. C : A significant cell death was observed between the 5.0 mM bacitracin and controls group after 3 days. however, 1.25, 2.5, and 3.75 mM bacitracin did not induced significant more cell death. D : A similar result was detected on day 4. *p<0.05, ***p<0.001.

• 

  Fig. 3 Bacitracin interferences the integrin function in respect to cell adhesion and morphology. A : Adhesion assay shows that the bacitracin treated and non-treated U87-MG cells exhibit different adhesion ability at the treatment doses and times, and each data point represents the mean+SD of 5 groups. B : Morphology assay shows that the cell morphology is altered after treatment with bacitracin at the concentrations of 2.5 and 3.75 mM (100×magnification). The image with red border in the 3.75 mM group, indicate the effect of bacitracin in cell morphology is reversible. ***p<0.001.

• 

  Fig. 4 Bacitracin inhibits U87-MG cell migration through affects the expression of certain molecules. A : The images show the initial and final stages of 4 groups (400×magnification). The chart show the moving tendency of U87-MG cells under different conditions. B : Western blot with labeled PDI on the cell surface shows that there is no remarkable distinction in total PDI on the cell surface; conversely, reduced PDI is increased along with an increase of the concentration of bacitracin. At the top figure the two bands indicate the members of PDI family. C : The p-FAK and MMP-2 expression decrease in the treated U87-MG cells, compare with the control group; however, the expression of PDI shows no significant difference under the same condition. In the MMP-2 image, the upper bands and the lower bands indicate latent form and activate form, respectively. D : The expression of MMP-2 is decreased with increasing of bacitracin concentration as shown by RT-PCR. E : The gelatin zymography shows that the expression of secreted MMP-2 decreases in a dose-dependent manner. The upper and lower bands also indicate the latent form and activate form, respectively. PDI : protein disulfide isomerase, p-FAK : phosphorylated focal adhesion kinase, MMP-2 : matrix metalloproteinase-2.

• 

  Fig. 5 Bacitracin inhibits U87-MG cell invasion in brain-slice. A : The cells radiate out in all direction and exhibit a high invasiveness. B : The cells cultured in the media contained 2.5 mM bacitracin, aggregate in the hole of the brain-slice. Although the cells show a high density, they always exhibit a low invasiveness, compare with control group. All the images are obtained by confocal laser scanning microscopy (100×magnification).

• 

  Fig. 6 The expression of PDI in human tissue samples. A : Immunohistochemistry of a glioblastoma sample obtained from a patient show the PDI location in comparison between the tumor core and the tumor margin. Left : Low magnification human glioblastoma immunohistochemistry. Right : High magnification immunohistochemistry; black asterisk indicate the tumor core and low PDI expression; black arrows indicate tumor margin and high PDI expression. B : The expression of PDI shows by the Western blot, in WHO grade II, grade III and grade IV gliomas. The PDI expression in grade IV glioma showed a significant difference compare with that in gliomas of the other two grades *p<0.05, ***p<0.001. PDI : protein disulfide isomerase.

• 

  Fig. 7 Schematic model showing the potential role of bacitracin in inhibiting glioma cell invasion. Oxidized protein disulfide isomerase (PDI) and reduced PDI interconvert under the bacitracin-free condition. During the transformation process, PDI alters the conformation of integrin to a high affinity state and integrin binds tightly with the extracellular matrix (ECM) (integrin activation). Then, focal adhesion kinase (FAK), one of the downstream molecules of integrin, is phosphorylated, which increases matrix metalloproteinase-2 (MMP-2) production. Finally, the cell secretes MMP-2, resulting in increased cell invasion. The PDI cycle was disturbed after the cells were treated with bacitracin. Bacitracin binding with reduced PDI led to lack of oxidation of reduced PDI. Only reduced PDI was present on the cell membrane after extending the bacitracin treatment time. Then, integrin was not activated, resulting in decreased FAK phosphorylation and MMP-2 production. Finally, MMP-2 secretion decreased, which disturbed cell invasion.

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