Suppression of matrix metalloproteinase activity by SI-27: detection by a new activity assay with S-2444, a specific chromogenic peptide.
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.
Yoshida
Daizo
D
Department of Neurosurgery, Nippon Medical School, Tokyo, Japan. dyoshida@nms.ac.jp
Watanabe
Kunihiro
K
Noha
Masahiro
M
Takahashi
Hiroshi
H
Teramoto
Akira
A
D002863:Chromogenic Compounds; D003094:Collagen / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005910:Glioma / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000008:administration & dosage / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002863:Chromogenic Compounds; D007527:Isoenzymes; D061965:Matrix Metalloproteinase Inhibitors; D009842:Oligopeptides; D012333:RNA, Messenger; C421267:SI 27; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2; C028190:5-oxo-prolyl-glycyl-arginine-4-nitroanilide; D003094:Collagen; D020782:Matrix Metalloproteinases