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 antica rcinogenic agents in in vivo and in vitro systems have recently been r eviewed. These studies suggest that the protease inhibitors which prev ent carcinogenesis affect processes in the early stages of carcinogene sis, although they can be effective at long time periods after carcino gen exposure in both in vitro and in vivo systems. While there is stro ng evidence that these protease inhibitors can affect both the initiat ion and promotion stages of carcinogenesis, they have no effect on alr eady transformed cells. Our results have suggested that the first even t in carcinogenesis is a high frequency epigenetic event and that a la ter event, presumably genetic, leads to the malignant state. Protease inhibitors appear capable of reversing the initiating event, presumabl y by stopping an ongoing cellular process begun by carcinogen exposure . The major lines of investigation on the mechanism of the protease in hibitor suppression of carcinogenesis relate to the ability of anticar cinogenic protease inhibitors to affect the expression of certain onco genes, and the levels of certain types of proteolytic activities. The anticarcinogenic protease inhibitors have no observable effects on nor mal cells, but can reverse carcinogen-induced cellular changes for sev eral different end-points studied. The most direct method of deternini ng the mechanism of action of the anticarcinogenic protease inhibitors is to identify and characterize the proteases with which they interac t. 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. Pr oteases have been identified by both substrate hydrolysis and affinity chromatography. Using substrate hydrolysis, we examined the ability o f cell homogenates to cleave specific substrates and then determined t he ability of various protease inhibitors to affect that hydrolyzing a ctivity. Affinity chromatography can isolate specific proteases that d irectly interact with anticarcinogenic protease inhibitors. As example s, the Boc-Val-Pro-Arg-MCA hydrolyzing activity was identified by subs trate hydrolysis, and a 43 kDa protease has been identified by affinit y chromatography. The isolation and characterization of these protease s has been and will continue to be a subject of investigation in our l aboratory. Our studies on anticarcinogenic protease inhibitors have su ggested 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 concentrat e (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 o rigin; when given to animals by several different routes of administra tion (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 [1]. 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 fro m the FDA. In human trials, elevated levels of proteolytic activities known to be affected by BET serve as intermediate marker end-points (I ME) in the cells of tissues having premalignant characteristics or whi ch are known to be at higher-than-normal risks of cancer development. fn previous animal studies, BBI was capable of bringing such elevated levels of proteolytic activity back to normal levels in the normal-app earing areas of carcinogen-treated tissue. We have recently discovered that BBI/BBIC can increase the levels of our marker proteolytic activ ities in premalignant cells and tissues, which could be highly relevan t to the mechanisms of action of the anticarcinogenic protease inhibit ors. These findings are summarized here. (C) 1995 Wiley-Liss, Inc.