Osthole suppresses the migratory ability of human glioblastoma multiforme cells via inhibition of focal adhesion kinase-mediated matrix metalloproteinase-13 expression

Abstract

Glioblastoma multiforme (GBM) is the most common type of primary and malignant tumor occurring in the adult central nervous system. GBM often invades surrounding regions of the brain during its early stages, making successful treatment difficult. Osthole, an active constituent isolated from the dried C. monnieri fruit, has been shown to suppress tumor migration and invasion. However, the effects of osthole in human GBM are largely unknown. Focal adhesion kinase (FAK) is important for the metastasis of cancer cells. Results from this study show that osthole can not only induce cell death but also inhibit phosphorylation of FAK in human GBM cells. Results from this study show that incubating GBM cells with osthole reduces matrix metalloproteinase (MMP)-13 expression and cell motility, as assessed by cell transwell and wound healing assays. This study also provides evidence supporting the potential of osthole in reducing FAK activation, MMP-13 expression, and cell motility in human GBM cells.

Figure 1.

Inhibitory effects of osthole on the proliferation of human glioma cells. Cell proliferation of two different human glioma cells (U251 and HS683) is shown. Cells were incubated with various concentrations of osthole (1, 10, or 30 μM) or vehicle for 24 h and the rate of inhibition was determined by (A) MTT assay and (B) SRB assay. Results are expressed as the means ± S.E.M. of at least three independent experiments. * p < 0.05 compared with the vehicle treatment group.

Figure 2.

Osthole inhibits migration activity of human glioma cells. By using a cell culture insert system, in vitro migration activities were examined. (A) After incubating cells with various concentrations of osthole (1, 10, or 30 μM) or vehicle for 24 h, we found that osthole inhibited migration activity in U251 and HS683 cells. Results are expressed as means ± S.E.M. of at least three independent experiments; (B) Cells were treated with various concentrations of osthole or vehicle for 24 h, and migrating cells were visualized by phase-contrast imaging. Results are expressed as means ± S.E.M. of at least three independent experiments. * p < 0.05 compared with control group.

Figure 3.

Osthole inhibits human glioma cells motility. Cells were seeded on the migration insert for 24 h and treated with various concentrations of osthole (1, 10, or 30 μM) or vehicle for another 16 h. Migrating cells were identified by wound-healing assay and visualized by phase-contrast imaging. We found that osthole inhibited cells motility in (A) U251 and (B) HS683 cells. Results are expressed as means ± S.E.M. of at least three independent experiments. * p < 0.05 compared with control group.

Figure 4.

Osthole-directed migration activity involves down-regulation of MMP-13 and cell motility-dependent FAK in human glioma cells. Cells were incubated with various concentrations of osthole (1, 10, or 30 μM) or vehicle for 24 h, after which the supernatant and cell lysate extracts were collected from U251 (A) and HS683 (B) cells. MMP-13 enzymatic activities were determined by gelatin zymography (A and B); MMP-13 protein levels were determined by western blot (C and D); and phosphorylated FAK was determined by western blot analysis (E and F). Results are expressed as means ± S.E.M. of at least three independent experiments. * p < 0.05 compared with control group.

Figure 5.

Down-regulation of osthole in migration-prone human glioma cells. (A) After 10 rounds of selection of U251 and HS683 cells using a cell culture insert system, the migration-prone subline (P10) exhibited higher migration ability than the original U251 and HS683 cells. Results are expressed as means ± S.E.M. of three independent experiments. * p < 0.05 compared with the original group (P0); (B) After incubating the migration-prone subline (P10) of U251 and HS683 cells with various concentrations of osthole (10 or 30 μM) or vehicle for 24 h, we found that osthole inhibited migration-prone subline (P10) migration activity in U251 and HS683 cells. Results are expressed as means ± S.E.M. of at least three independent experiments; (C and D). The migration-prone subline (P10) were seeded for 24 h and treated with various concentrations of osthole (10 or 30 μM) or vehicle for another 16 h. The cells migration were determined by wound-healing assay and visualized by phase-contrast imaging. Results are expressed as means ± S.E.M. of at least three independent experiments. * p < 0.05 compared with control group.

Figure 6.

Osthole-directed migration activity involves down-regulation of MMP-13 and cell motility dependent FAK in migration-prone human glioma cells. The migration-prone subline (P10) was incubated with various concentrations of osthole (10 or 30 μM) or vehicle for 24 h, after which the supernatant and cell lysate extracts were collected in P10 cells from U251 and HS683. MMP-13 enzymatic activities were determined by gelatin zymography (A and B); MMP-13 protein levels were determined by western blot (C and D); and phosphorylated FAK was determined by western blot analysis (E and F). Results are expressed as means ± S.E.M. of at least three independent experiments. * p < 0.05 compared with control group.