Effects of protease inhibitors on levels of proteolytic activity in normal and premalignant cells and tissues.

Our studies utilizing different types of protease inhibitors as anticarcinogenic agents in in vivoand in vitro systems have recently been reviewed. These studies suggest that the protease inhibitors which prevent carcinogenesis affect processes in the early stages of carcinogenesis, although they can be effective at long time periods after carcinogen exposure in both in vitro and in vivo systems. While there is strong evidence that these protease inhibitors can affect both the initiation and promotion stages of carcinogenesis, they have no effect on already transformed cells. Our results have suggested that the first event in carcinogenesis is a high frequency epigenetic event and that a later event, presumably genetic, leads to the malignant state. Protease inhibitors appear capable of reversing the initiating event, presumably by stopping an ongoing cellular process begun by carcinogen exposure. The major line of investigation on the mechanism of the protease inhibitor suppression of carcinogenesis relate to the ability of anticarcinogenic protease inhibitors to affect the expression of certain oncogenes, and the levels of certain types of proteolytic activities. The anticarcinogenic protease inhibitors have no observable effects on normal cells, but can reverse carcinogen-induced cellular changes for several different end-points studied. The most direct method of determining the mechanism of action of the anticarcinogenic protease inhibitors is to identify and characterize the proteases with which they interact. In the cells of the in vivo and in vitro systems in which protease inhibitors can prevent carcinogenesis, only a few proteases have been observed to interact with the anticarcinogenic protease inhibitors. Proteases have been identified by both substrate hydrolysis and affinity chromatography. Using substrate hydrolysis, we examined the ability of cell homogenates to cleave specific substrates and then determined the ability of various protease inhibitors to affect that hydrolyzing activity. Affinity chromatography can isolate specific proteases thatb directly interact with anticarcinogenic protease inhibitors. As examples, the Boc-Val-Pro-Arg-MCA hydrolyzing activity was identified by substrate hydrolysis, and a 43 kDa protease has been identified by affinity chromatography. The isolation and charactetrization of these proteases has been and will continue to be a subject of investigation on our laboratory. Our studies on anticarcinogenic protease inhibitors have suggested that the Bowman-Birk Inhibitor (BBI) derived from soybeans is a particularly effective anticarcinogenic protease inhibitor. BBI has been studied both as a pure protease inhibitor, or purified BBI (PBBI), and as an extract of soybeans enriched in BBI, termed BBI concentrate (BBIC). PBBI and/or BBIC have been shown to suppress carcinogenesis in three different species (mice, rats and hamsters); in several organ systems/tissue types (colon, liver, lung, esophagus and cheek pouch [oral epithelium]); in cells of both epithelial and connective tissue origin; when given to animals by severasl different routes of administration (including the diet); leading to different types of cancer (e.g., squamous cell carcinomas, adenocarcinomas, angiosarcomas, etc.), and induced by a wide variety of chemical and physical carcinogens [l]. We originally identified BBI as an anticarcinogenic agent in an in vitro transformation assay system. BBI, as BBIC, has recently risen to the human trial stage and has achieved Investigational New Drug status from the FDA. In human trials, elevated levels of proteolytic activities known to be affected by BBI serve as intermediate marker endpoints (IME) in the cells of tissues having premalignant characteristics or which are known to be at higher-than-normal risks of cancer development. In previous animal studies, BBI was capable of bringing such elevated levels of proteolytic activity back to normal levels in the normal-appearing areas of carcinogen-treated tissue. We have recently discovered that BBI/BBIC can increase the levels of our marker proteolytic activities in premalignant cells and tissues, which could be highly relevant to the mechanisms of action of the anticarcinogenic protease inhibitors. These findings are summarized here.