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Abstract
Glioblastoma multiforme is one of the most common and aggressive tumors in central nervous system. It often possesses characteristic necrotic lesions with hemorrhages, which increase the chances of exposure to thrombin. Thrombin has been known as a regulator of MMP-9 expression and cancer cell migration. However, the effects of thrombin on glioma cells have not been clearly understood. In the present study, influences of thrombin on glioma cell migration were examined using Boyden chamber migration assay and thrombin-induced changes in MMP-9 expression were measured using zymography, semi-quantitative RT-PCR, and Western blotting. Furthermore, underlying signaling pathways by which thrombin induces MMP-9 expression were examined. Thrombin-induced migration and MMP-9 expression were significantly potentiated in the presence of wortmannin, a PI3K inhibitor, whereas MAPK inhibitors suppressed thrombin-induced migration and MMP-9 expression in C6 glioma cells. The present data strongly demonstrate that MAPK and PI3K pathways evidently regulate thrombin-induced migration and MMP-9 expression of C6 glioma cells. Therefore, the control of these pathways might be a beneficial therapeutic strategy for treatment of invasive glioblastoma multiforme.
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Fig. 1.
Thrombin stimulates C6 glioma cell migration. (A) Representative images of C6 cell migration assay. Thrombin (25 U/ml) treatment for 6 hr exhibited approximately 2 fold increase in number of migrating cells. Migrating cells and unmigrating cells were separated by polyethylene terephthalate (PET) membranes. Microscopy images were detected the migrating cells on the lower surface of the membrane. (B) Quantitative analysis of migration assay. The cell migration was quantified by counting the cells that migrating through the membranes. Cell counting data were expressed as a percentage in comparison with control group. The experiments were conducted in triplicate and data are shown as the means±SD. ∗∗p<0.01 indicates statistically significant difference with control.
Fig. 2.
MAPK inhibitors (MAPKIs) inhibit thrombin-induced C6 glioma cell migration. (A) Representative images of the MAPKI effects on thrombin-induced migration. SB203580, SP600125, and U0126 were pretreated for 1 hr at the concentrations of 20, 20, and 10μM, respectively, followed by thrombin treatment for 6 hr. (B) Quantitative analysis of migration assay. The cell migration was quantified by counting the cells that migrating through the membranes. All data were obtained from three independent experiments and presented as the means±SD. ∗p<0.05 indicates statistically significant difference with control. #p<0.05 and ##p<0.01 indicate statistically signiticunt difference with thrombin.
Fig. 3.
Thrombin-induced chemotactic migration was markedly increased by PI3K inhibitor, wortmannin. (A) Potentiating effects were observed in the group that treated thrombin for 6 hr subsequent to preincubated with wortmannin for 1 hr. (B) Quantitative analysis of migration assay. The cell migration was quantified by counting the cells that migrating through the membranes. All data were obtained from three independent experiments and presented as the means±SD. ∗p<0.05 and ∗∗p<0.01 indicate statisticodly significant difference with control. #p<0.05 indicates statistically significant difference between thrombin and wortmannin thrombin.
Fig. 4.
Effects of thrombin and MAPK or PI3K inhibitors treatment on MMP-9 expression in protein level. All inhibitors were pretreated for 1 hr at the indicated concentrations followed by 24 hr of thrombin (5 U/ml) treatment: SB203580 and SP600125, 20 μM; U0126 and wortmannin, 10 μM. C6 cells were incubated with inhibitors in the serum free media. (A) MMP-9 activities were analyzed by zymography. Thrombin-induced MMP-9 expression was blocked by MAPK inhibitors and increased by PI3K inhibitor. (B) The cellular level of MMP-9 was examined by western blot analysis. β-actin was used as an internal control. ∗p<0.05 indicates statistically signiticant difference with control. #p<0.05 and ##p<0.01 indicate statistically signiticant difference with thrombin.
Fig. 5.
Effects of thrombin and MAPK or PI3K inhibitors treatment on MMP-9 expression in mRNA level. C6 cells were pretreated with inhibitors for 1 hr, followed by thrombin (5 U/ml) treatment for 4 hr. Subsequently, RNA samples were isolated and subjected to semi-quantitative RT-PCR for MMP-9 with a housekeeping gene, GAPDH, as an internal control. MMP-9 mRNA expression patterns were similar to western blot and zymographic data.
Fig. 6.
Thrombin-induced migration was directly blocked by batimastat, an MMP-9 inhibitor (MMPI). (A) Representative images of the effect of MMPI on thrombin-induced C6 cell migration. 0.5μM of batimastat was pretreated for 1 hr and thrombin was subsequently treated for 6 hr. (B) Quantitative analysis of migration assay. The cell migration was quantified by counting the cells that migrating through the membranes. All data were obtained from three independent experiments and presented as the means±SD. ∗∗p<0.01 and #p<0.05 indicate statistically signficant difference between control and thrombin, and thrombin and MMPI thrombin, respectively.
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