Abstract
Proteases facilitate several steps in cancer progression. To identify proteases most suitable for drug targeting, actual enzyme activity and not messenger RNA levels or immunoassay of protein is the ideal assay readout. An automated microtiter plate assay format was modified to allow detection of all four major classes of proteases in tissue samples. Fifteen sets of colorectal carcinoma biopsies representing primary tumor, adjacent normal colon, and liver metastases were screened for protease activity. The major proteases detected were matrix metalloproteases (MMP9, MMP2, and MMP1), cathepsin B, cathepsin D, and the mast cell serine proteases, tryptase and chymase. Matrix metalloproteases were expressed at higher levels in the primary tumor than in adjacent normal tissue. The mast cell proteases, in contrast, were at very high levels in adjacent normal tissue, and not detectable in the metastases. Cathepsin B activity was significantly higher in the primary tumor, and highest in the metastases. The major proteases detected by activity assays were then localized in biopsy sections by immunohistochemistry. Mast cell proteases were abundant in adjacent normal tissue, because of infiltration of the lamina propria by mast cells. Matrix metalloproteases were localized to the tumor cells themselves; whereas, cathepsin B was predominantly expressed by macrophages at the leading edge of invading tumors. Although only low levels of urinary plasminogen activator were detected by direct enzyme assay, immunohistochemistry showed abundant protein within the tumor. This analysis, surveying all major classes of proteases by assays of activity rather than immunolocalization or in situ hybridization alone, serves to identify proteases whose activity is not completely balanced by endogenous inhibitors and which may be essential for tumor progression. These proteases are logical targets for initial efforts to produce low molecular weight protease inhibitors as potential chemotherapy.
Highlights
Studies that detect key biochemical differences between tumor cells and their normal counterparts are rapidly progressing from traditional cell culture studies to direct analysis of tumor and normal tissue from animal models or human specimens
Frozen tissue samples from time-matched colon cancers sets (tumor, adjacent (5 cm) normal tissue, and liver metastases stored at Ϫ70ЊC), were homogenized in 250 l/mg ice cold phosphate-buffered saline (PBS) with 0.1% Triton X-100 using a Tenbrock homogenizer on ice
Major Proteases Detected in Colon Biopsies and Correlation with Tumor Progression
Summary
Studies that detect key biochemical differences between tumor cells and their normal counterparts are rapidly progressing from traditional cell culture studies to direct analysis of tumor and normal tissue from animal models or human specimens. Matrix metalloproteases were expressed at higher levels in the primary tumor than in adjacent normal tissue. Only low levels of urinary plasminogen activator were detected by direct enzyme assay, immunohistochemistry showed abundant protein within the tumor. Conclusions: This analysis, surveying all major classes of proteases by assays of activity rather than immunolocalization or in situ hybridization alone, serves to identify proteases whose activity is not completely balanced by endogenous inhibitors and which may be essential for tumor progression. These proteases are logical targets for initial efforts to produce low molecular weight protease inhibitors as potential chemotherapy
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