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Updated: Sep 30, 2026
Detection of Functional Matrix Metalloproteinases by Zymography
Published on: November 8, 2010
Suppression of matrix metalloproteinase activity by SI-27: detection by a new activity assay with S-2444, a specific
Daizo Yoshida1, Kunihiro Watanabe, Masahiro Noha
1Department of Neurosurgery, Nippon Medical School, Tokyo, Japan. dyoshida@nms.ac.jp
Abstract:
We have previously reported on the anti-invasive and angiosuppressive effects of SI-27, an anti-matrix metalloproteinase (MMP) agent. The molecular mechanism of its anti-MMP action, however, has not yet been determined. The purpose of this study was to investigate the effects of SI-27 on MMP- 1, -2, -3, -9, and TIMP-1, -2 secreted by human glioma cell lines (U87MG, U251MG, U373MG, and Y98G). When cells were exposed to non-cytotoxic concentrations of SI-27 (preliminarily determined by the MTT assay), expressions of mRNAs for the enzymes was not inhibited. For an MMP activity assay, we employed the fact that active MMPs could cleave modified pro-urokinase to form active urokinase, which then acted on S-2444 peptide to create a chromogenic product. Secretion of all pro-MMPs from glioma cells was not significantly reduced by SI-27. However, activation of pro-MMPs was significantly inhibited in a dose-dependent manner ((IC50 values for MMP-2; U87MG, 3.5 microg/ml; U25 IMG, 4.2 microg/ml; U373MG, 4.8 microg/ml; Y98G, 4.0 degreesg/ml); (IC50 values for MMP-9; 251MG, 7.2 microg/ml, U373MG, 2.8 microg/ml). In addition, active MMPs were not inhibited by SI-27. These findings were supported by zymographic analysis and by collagenolysis assay data. TIMP-1 and -2 were also not inactivated by SI-27. These findings suggest that SI-27 targets the activation process of pro-MMP. S-2444, a specific chromogenic peptide, was useful for quantitative analysis of the activity of MMP subtypes in this study.
Insights
SI-27, an anti-matrix metalloproteinase (MMP) agent, inhibits pro-MMP activation in glioma cells without affecting MMP expression or activity. This study reveals SI-27
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Matrix metalloproteinases (MMPs) are crucial in cancer invasion and angiogenesis.
- SI-27, an MMP inhibitor, has demonstrated anti-invasive and anti-angiogenic effects.
- The precise molecular mechanism of SI-27's anti-MMP action remains undetermined.
Purpose of the Study:
- To elucidate the molecular mechanism of SI-27's anti-MMP activity.
- To investigate the effects of SI-27 on MMP-1, -2, -3, -9, and TIMP-1, -2 expression and activity in human glioma cell lines.
- To determine if SI-27 inhibits MMP secretion, activation, or activity.
Main Methods:
- Human glioma cell lines (U87MG, U251MG, U373MG, Y98G) were treated with non-cytotoxic concentrations of SI-27.
- Messenger RNA (mRNA) expression of MMPs was assessed.
- MMP activity was measured using a chromogenic assay involving pro-urokinase activation and S-2444 peptide cleavage.
- Zymographic analysis and collagenolysis assays were performed.
- Tissue inhibitors of metalloproteinases (TIMP-1, -2) inactivation was evaluated.
Main Results:
- SI-27 did not inhibit the expression of MMP mRNAs or the secretion of pro-MMPs.
- SI-27 significantly inhibited the activation of pro-MMPs in a dose-dependent manner, with specific IC50 values for MMP-2 and MMP-9.
- SI-27 did not inhibit the activity of already active MMPs.
- TIMP-1 and TIMP-2 remained active in the presence of SI-27.
- Zymography and collagenolysis assays corroborated the inhibition of MMP activation.
Conclusions:
- SI-27's anti-MMP mechanism involves the inhibition of pro-MMP activation, not MMP expression or activity.
- This targeted inhibition of MMP activation represents a novel therapeutic strategy for glioma.
- The chromogenic peptide S-2444 is a valuable tool for quantifying MMP subtype activity.
