Esculetin, a potent non-competitive inhibitor of lipoxygenase, has been shown to inhibit vascular smooth muscle cell (VSMC) proliferation. However, the effect of esculetin on the matrix metalloproteinase-9 (MMP-9) regulation responsible for cell migration and invasion has not been previously investigated. The results of the present study showed the esculetin (12.5-25 µg/ml) induced the inhibition of migration and invasion in tumor necrosis factor-α (TNF-α)-treated VSMC, as demonstrated by a matrigel invasion assay and wound healing analysis. However, esculetin did not affect cell viability in TNF-α-treated VSMC under 0-25 µg/ml concentration conditions. In addition, both zymographic and immunoblot experiments showed that esculetin suppressed the TNF-α-induced expression of MMP-9 in VSMC in a dose-dependent manner. Furthermore, the treatment of cells with esculetin decreased the activity of the TNF-α-induced MMP-9 promoter, which was driven by a luciferase reporter. Finally, esculetin reduced the binding activities of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), which are cis-elements present in the promoter of the MMP-9 gene, in TNF-α-treated VSMC. Taken together, these results demonstrated that esculetin decreased the migration and invasion of cells by suppressing MMP-9 expression, which subsequently reduced the binding activities of NF-κB and AP-1 in TNF-α-treated VSMC. These novel findings provide basic information for effective therapeutic treatment with esculetin for atherosclerotic disease.

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