Heterogeneous suppression of experimentally induced colon cancer metastasis in rat liver lobes by inhibition of extracellular cathepsin B.

Metastatic rat colon cancer cells but not normal rat hepatocytes showed activity of cathepsin B on their plasma membranes. Activity was visualized in living cells with a new fluorogenic substrate, [Z-Arg]2-cresyl violet, and confocal microscopy. When these cancer cells were injected into the portal vein of rats, the animals developed tumors in the liver in a heterogeneous fashion. Three- to four-fold more tumors were found in the small caudate lobe than in the other three large lobes of the liver. Oral treatment with a selective water-soluble inhibitor of extracellular cathepsin B, Mu-Phe-homoPhe-fluoromethylketone, resulted in 60% reduction of the number of tumors and 80% reduction of the volume of tumors in the three large lobes whereas tumor development was not affected in the small caudate lobe. This study supports the conclusions that (a) extracellular cathepsin B plays a crucial but complex role in liver colonisation by rat colon carcinoma cells in vivo, (b) its selective inhibition suppresses tumor growth heterogeneously in the liver and (c) the caudate lobe of the liver is a relatively large risk factor for tumor development.

Ala-Pro-cresyl violet, a synthetic fluorogenic substrate for the analysis of kinetic parameters of dipeptidyl peptidase IV (CD26) in individual living rat hepatocytes.

A new type of fluorogenic substrates for proteases based on the leaving group cresyl violet has been synthesized. Cresyl violet is not fluorescent when amino acids or peptide groups are attached but becomes highly fluorescent after proteolytic liberation. Its fluorescence shows linearity with concentration and barely any fading. The properties of Ala-Pro-cresyl violet as substrate for dipeptidyl peptidase IV (DPPIV) (CD26) for localization and quantification of its activity in individual freshly isolated living rat hepatocytes were investigated using confocal microscopy, image analysis, and flow cytometry. DPPIV activity was localized exclusively in patches at plasma membranes likely being bile canalicular domains. Activity was analyzed quantitatively in individual cells by capturing series of images in time. Production of fluorescence was analyzed on the basis of the series of digital images and it appeared to be nonlinear with time. By calculation of the initial velocity at time zero, activity of DPPIV per individual hepatocyte was calculated. Cresyl violet-dependent fluorescence appeared in a similar way when cells were analyzed by flow cytometry. A dipeptide phosphonate inhibitor inhibited production of fluorescence competitively with a Ki of 7 microM. K(m) values in individual hepatocytes varied in the range of 6-22 microM depending on the individual rat from which the hepatocytes were obtained, whereas the Vmax varied in the range of 4-16 nU. K(m) and Vmax values per individual rat were inversely correlated indicating posttranslational regulation of the kinetic parameters of DPPIV. This relationship was lost when membrane fractions of the same hepatocyte suspensions were analyzed. It is concluded that cresyl violet-based protease substrates are the compounds of choice to localize and quantify protease activity in living cells and tissues.