The metastasis associated 72-kDa type IV collagenase is secreted as a latent proenzyme which is converted to an active 62-kDa form by autoproteolytic removal of an amino terminal profragment. The region immediately upstream from the cleavage site contains a highly conserved peptide sequence, MRKPRCGNPDV, which is present in all known members of the matrix metalloproteinase family. Evidence implicates the cysteine residue of this sequence as critical for maintenance of the latent form through coordination with the catalytic zinc atom of the active site. A synthetic peptide, TMRKPRCGNPDVAN (peptide 74), encompassing this conserved sequence, has been shown to inhibit the activated form of the 72-kDa type IV collagenase in vitro. In the present study we examine the ability of this peptide inhibitor to modulate tumor cell invasiveness. Peptide 74 and the control peptide 78, which contains a single substitution of serine for the “critical” cysteine residue, were added at 30 µm concentrations to the upper compartment of the Boyden chamber in the chemoinvasion assay using HT1080 and A2058 human tumor cells. In this assay a layer of reconstituted basement membrane, Matrigel, is coated onto chemotaxis filters and acts as a barrier to the migration of cells in the Boyden chambers. Only cells with invasive capacity can cross the Matrigel barrier. Peptide 74 containing the cysteine residue inhibited the invasion of both the HT1080 and A2058 cells through the Matrigel barrier, control peptide 78 was not inhibitory. Both peptides were shown to be without cytotoxic action and did not inhibit chemotaxis or affect cell number. This study demonstrates that addition of an excess peptide containing the matrix metalloproteinase prosegment inhibitory sequence can inhibit invasive activity at the cellular level and suggests that this may be a useful strategy to modulate tumor cell invasiveness in vivo.
Research was supported by grants from the Consiglio Nazionale delle Ricerche (BTBS IN.91.01269.PF70 to A. A.) and Associazione Italiana Ricerca sul Cancro.