Suppression of in vitro invasion of human fibrosarcoma cells by a leupeptin analogue inhibiting the urokinase-plasmin system.

A leupeptin analogue, pyroglutamyl-Leu-Arg-CHO (Pyr-Leu-Arg-CHO), is an inhibitor of urokinase and plasmin, while leupeptin inhibits only plasmin. Pyr-Leu-Arg-CHO was shown to inhibit in vitro invasion of human fibrosarcoma HT1080 cells reducing cellular collagenase activity. Pyr-Leu-Arg-CHO suppressed the invasion of the cells in a Boyden chamber assay with an IC50 of 12 micrograms/ml. Addition of plasminogen to HT1080 cells increased the type IV collagenase activity, and Pyr-Leu-Arg-CHO inhibited this activation of the collagenase with an IC50 of 3 micrograms/ml. Leupeptin inhibited both the invasion and collagenase activation at higher concentrations than that of Pyr-Leu-Arg-CHO. The gelatin zymography of the conditioned medium revealed that a new gelatinolytic band, possibly an activated form of MMP-2, appeared by the addition of plasminogen. The activation of MMP-2 was also inhibited strongly by Pyr-Leu-Arg-CHO. These results indicate that Pyr-Leu-Arg-CHO suppresses the in vitro invasion by preventing the activation of type IV collagenase through inhibition of the urokinase-plasmin system.