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Selective Inhibition of Proteolytic Enzymes in an in Vivo Mouse Model for Experimental Metastasis¹

Department of Biochemistry and Biophysics, Stritch School of Medicine, Loyola University of Chicago, Maywood, Illinois 60153

Peptide aldehyde transition state analogue inhibitors of serine and cysteine proteases have been used to selectively inhibit proteases for which prior evidence supports a role in tumor cell metastasis. These enzymes include cathepsin B, urokinase plasminogen activator (PA), and thrombin. The inhibition constants of the peptidyl aldehyde inhibitors show that they are highly selective for a particular targeted serine or cysteine protease. The inhibitors are introduced by i.p. injection or by miniosmotic pumps into syngeneic C57BL/6 mice also given injections of B16-F10 melanoma cells, and the number of metastatic foci in the lung was determined. While the injection protocol gave an initially high but changing in vivo concentration of inhibitor over time, the minipump implant gave a constant steady state concentration of inhibitor over 5–7 days. Minipump infusion of leupeptin (acetylleucylleucylargininal), a strong inhibitor of cathepsin B at a steady state plasma concentration 1000-fold greater than its K_{i(cathepsin B)}, gave no significant decrease in lung colonization by the B16 tumor cells. Ep475, a stoichiometric irreversible peptide inhibitor of cathepsin B-like proteases, also did not significantly inhibit metastatic foci formation. Introduction of selective inhibitors of urokinase PA, tert-butyloxycarbonylglutamylglycylargininal and H-glutamylglycylargininal at concentrations near its K_i, produced no significant decrease in mouse lung colonization. The selective thrombin inhibitor D-phenylalanylprolylargininal infused to a steady state concentration 100-fold greater than its K₁ dramatically increased B16 melanoma colonization of mouse lung. The results indicate that neither secreted cathepsin B-like nor urokinase PA have roles in B16 colonization of mouse lung, while thrombin may have a role in preventing metastasis. These experiments do not eliminate roles for a cathepsin B-like enzyme or urokinase PA in the initial steps of the metastatic process.

¹ Research Support from the National Cancer Institute (Grant CA-43530) and the Potts Endowment Fund is gratefully acknowledged.

² Research comprises a part of a Dissertation to be submitted in partial fulfillment of the requirements for the Ph.D. degree at Department of Biochemistry and Biophysics, Loyola University of Chicago.

³ To whom requests for reprints should be addressed.

Received 11/13/85. Revised 3/16/86. Accepted 5/12/86.

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