Mechanisms of Cancer Prevention: Effects of Protease Inhibitors on Proteases and Gene Expression
Our studies utilizing different Types of protease inhibitors as anticarcinogenic agents in in vivo and in vitro systems have recently been reviewed (1). These studies suggest that the protease inhibitors which prevent carcinogenesis are affecting 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 cells which are already transformed. 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 to be capable of reversing the initiating event, presumably by stopping an ongoing cellular process begun by carcinogen exposure. The major lines of investigation on the mechanism of the protease inhibitor suppression of carcinogenesis relate to the ability of anticarcinogenic protease inhibitors to affect: 1) the expression of certain oncogenes, and 2) the levels of certain Types of proteolytic activities. The anticarcinogenic protease inhibitors have no observable effects on normal cells, but have the ability to reverse carcinogen-induced cellular changes for several different endpoints 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 we’ve observed that 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. Utilizing substrate hydrolysis, we have examined the ability of cell homogenates to cleave specific substrates and have then determined the ability of various protease inhibitors to affect that hydrolyzing activity. Utilizing affinity chromatography, specific proteases directly interacting with the anticarcinogenic protease inhibitors can be identified. The Boc-Val-Pro-Arg-MCA hydrolyzing activity was identified by substrate hydrolysis and a 43 kDa protease has been identified by affinity chromatography. There is evidence that both of these different proteolytic activities are playing a role in the prevention of carcinogenesis by protease inhibitors.
Our studies on anticarcinogenic protease inhibitors have suggested that the Bowman-Birk Inhibitor (BBI), derived from soybeans, is a particularly effective anti-carcinogenic protease inhibitor. BBI has been studied both as a pure protease inhibitor, 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: 1) in three different species (mice, rats and hamsters), 2) in several organ systems/tissue Types (colon, liver, lung, esophagus and cheek pouch [oral epithelium]), 3) in cells of both epithelial and connective tissue origin, 4) when given to animals by several different routes of administration (including the diet), 5) leading to different Types of cancer (e.g., squamous cell carcinomas, adenocarcinomas, angiosarcomas, etc.), and 6) 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. In human trials, elevated levels of proteolytic activities known to be affected by BBI are serving as intermediate marker endpoints
human trials, elevated levels of proteolytic activities known to be affected by BBI are serving as intermediate marker endpoints (IME) in the cells of tissues at higher than normal risks of cancer development. In previous animal studies, we have observed that BBI is capable of bringing such elevated levels of proteolytic activity back to normal levels in normal appearing tissues. We have recently observed that BBI treatment can increase the levels of proteolytic activities in pre-malignant tissues. We hypothesize that the increased levels of proteolytic activities in pre-malignant cells and tissues are associated with a cell killing effect, leading to a degradation of the pre-malignant lesions. Although the mechanism for the anticarcinogenic effect(s) of BBI is unclear, BBI is a highly promising human cancer chemopreventive agent.
KeywordsProtease Inhibitor Proteolytic Activity Trypsin Inhibitory Activity Carcinogen Exposure Oral Carcinogenesis
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