Anticarcinogenic Protease Inhibitors Research Articles

Effects of the Bowman-Birk Inhibitor on Clonogenic Survival and Cisplatin- or Radiation-Induced Cytotoxicity in Human Breast, Cervical, and Head and Neck Cancer Cells

Bowman-Birk inhibitor (BBI) is a soybean-derived anticarcinogenic protease inhibitor previously shown to potentiate cisplatin-induced cytoxicity in human lung and ovarian cancer cells. To further assess the potential of BBI as a sensitizing agent for cancer radiotherapy and chemotherapy, we evaluated the effects of BBI and a soybean concentrate enriched in BBI known as BBI concentrate (BBIC) on clonogenic survival and radiation- or cisplatin-induced cell killing in MCF7 human breast carcinoma cells, SCC61 and SQ20B human head and neck carcinoma cells, HeLa, HeLa-R1, and HeLa-R3 human cervical carcinoma cells, MCF10 nontumorigenic human epithelial cells, HTori-3 nontumorigenic human thyroid epithelial cells, and C3H10T1/2 mouse fibroblast cells. BBI and BBIC significantly suppressed the clonogenic survival of MCF7 and SCC61 cells. BBIC also suppressed the survival of SQ20B cells and enhanced radiation-induced cell killing in SCC61 and SQ20B cells and cisplatin-induced cell killing in HeLa, HeLa-R1, and HeLa-R3 cells. In contrast, BBI and/or BBIC did not enhance radiation-induced cell killing in MCF10 cells or cisplatin-induced cell killing in C3H10T1/2 cells. BBI did not significantly affect the survival of SQ20B cells or enhance radiation-induced cell killing in SCC61 and SQ20B cells. The clonogenic survivals of MCF10 and C3H10T1/2 cells were not adversely affected by treatment with BBI or BBIC. The clonogenic survival of HTori-3 cells was only moderately suppressed by treatment with BBIC at ≥80 mg/ml. These results suggest that BBIC could be a useful agent for the potentiation of radiation- and cisplatin-mediated cancer treatment without significant adverse effects on surrounding normal tissues.

Effects of the Bowman-Birk protease inhibitor on survival of fibroblasts and cancer cells exposed to radiation and cis-platinum.

The Bowman-Birk inhibitor (BBI) is a soybean-derived anticarcinogenic protease inhibitor with anti-inflammatory activity. To assess the possibility of utilizing BBI for alleviating the side effects associated with lung cancer radiation and chemotherapy, we have determined the effects of BBI and a soybean concentrate enriched in BBI (known as BBIC) on radiation- and cis-platinum-induced cytotoxicity in A549 human lung cancer cells. The results demonstrated that neither BBI nor BBIC protected A549 cells from radiation- and cis-platinum-induced cytotoxicity. In fact, BBI and BBIC potentiated the cell-killing effects induced by cis-platinum alone, and BBIC treatment led to significantly enhanced cell killing by cis-platinum in combination with radiation treatment in the lung carcinoma cells. BBI conferred a significant protective effect onto mouse fibroblasts (10T1/2 cells) treated with cis-platinum in combination with 6 Gy of X-ray irradiation. These results suggest that BBI and BBIC, when given to lung cancer patients, are unlikely to interfere with cancer treatment utilizing radiation and cis-platinum.

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 vivo and 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 lines 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies related to the potential antigenicity of the Bowman-Birk inhibitor, an anticarcinogenic protease inhibitor isolated from soybeans.

Soybeans are known to contain certain proteins that are allergenic; therefore the anticarcinogenic Bowman-Birk protease inhibitor (BBI), an 8-kDa protein isolated from soybeans, could be capable of generating an immune response that would contraindicate its use as a cancer chemopreventive agent. In the present investigation, the stimulation of antibody production in response to BBI was measured after the administration of BBI by various routes and treatment regimens. A significant antibody titer to BBI was observed when the animals were treated with BBI by intraperitoneal injection once or three times per week. In these studies, higher antibody titers were observed at earlier time points. When the animals were treated with BBI by oral gavage, little or no antibody production was observed, regardless of the treatment regimen. The doses and treatment regimens used in this study have been previously demonstrated to be effective in preventing tumor formation. The results of this study indicate that antibody production in response to BBI administered orally should not present a complication to BBI treatment.

Potential mechanisms of antitumorigenesis by protease inhibitors.

The mechanisms by which protease inhibitors suppress carcinogenesis are unknown. From our studies, we believe 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 in carcinogenesis, even at long times after carcinogen exposure. Protease inhibitors are thought to stop an ongoing process begun by carcinogen exposure. Effects of protease inhibitors on the following phenomena are thought to be related to their anticarcinogenic activity: 1) ability to effect the expression of certain oncogenes, and 2) ability to return carcinogen-increased levels of certain proteolytic activities (e.g., Boc-val-pro-arg-MCA hydrolyzing activity) to normal levels. Other effects of anticarcinogenic protease inhibitors have also been observed; for example, we have reported that they can bring carcinogen-induced, elevated levels of gene amplification to nearly normal levels. The mechanism(s) by which protease inhibitors suppress carcinogenesis will not be determined with certainty until the mechanisms involved in cancer induction are known.

Intestinal epithelial cells contain a high molecular weight protease subject to inhibition by anticarcinogenic protease inhibitors

Protease inhibitors have been shown to be highly effective suppressors of carcinogenesis in both in vivo and in vitro model systems. For example, the soybean derived Bowman Birk inhibitor (BBI) has been shown to inhibit colon carcinogenesis. Although the precise mechanisms by which protease inhibitors suppress carcinogenesis are not known, it is believed that these compounds exert their anticarcinogenic effects by inhibiting specific cellular protease activities involved in the induction and/or expression of the transformed phenotype. In the current report, we describe a BBI-inhibitable proteolytic activity present in intestinal epithelial cells. The protease has a mass of ∼ 125 kDa, cleaves gelatin and will bind to a BBI-affinity resin. Subcellular fractionation experiments indicate that this protease is located in the 10 000 × g pellet (lysosomal/golgi fraction) of IEC17 cell homogenates. Further studies have revealed that this proteolytic activity is inhibited by BBI and DFP, but unaffected by EDTA, indicating that this enzyme is a serine protease. Our results suggest that the 125-kDa protease is a ‘target enzyme’ of the BBI in these cells.

Effects of activators and inhibitors of protein kinase C on X-ray induced malignant transformation in vitro

We have examined the effects on X-ray induced malignant transformation in vitro of a number of activators and inhibitors of protein kinase C (PKC). Several of these substances were found to enhance or inhibit transformation, and the extent of the effects on transformation were found to be consistent with the potencies of the substances in activating or inhibiting PKC. Additionally, the observed transformation enhancement was found to be reversed by the presence of the anticarcinogenic protease inhibitors antipain or the Bowman-Birk inhibitor. These results suggest that activation of protein kinase C may be involved in the mechanism of in vitro X-ray induced malignant transformation.

Is There a Critical Target Gene for the First Step in Carcinogenesis?

Our work has suggested that a high-frequency event is involved in the initiation phase of malignant transformation in vitro; a later, mutationlike event appears to be involved in the later stages of transformation. There may be no specific "target gene" which directly interacts with carcinogens. It is hypothesized that nonspecific types of DNA damage are involved in the induction of an ongoing process we know as carcinogenesis. Several genes could be involved in maintaining this process. Our recent results suggest that c-myc and c-fos could be involved in the early stages of carcinogenesis, as they are affected by anticarcinogenic protease inhibitors in a manner that corresponds to the way in which protease inhibitors suppress malignant transformation.

Identification of a proteolytic activity which responds to anticarcinogenic protease inhibitors in [formula omitted] cells

Untransformed and malignantly-transformed mouse embryo fibroblasts were found to contain an enzymatic activity which hydrolysed the synthetic substrate, Suc-Ala-Ala-Pro-Phe-AMC. This activity, of approximate molecular weight 55,000, which has been partially purified by ion-exchange and gel-filtration chromatography, was maximally active at neutral pH, associated with subcellular organelles or membranes and inhbited by EDTA, EGTA, phosphoramidon and 1,10 phenanthroline, but not by PMSF or pepstatin, indicating that it may be a metalloprotease. Several other protease inhibitors, such as chymostatin, TPCK and the BBI from soybean, were also potent inhibitors of the activity; these same inhibitors are known to suppress the malignant transformation of mouse embryo fibroblast cells in vitro induced by X-irradiation or chemical carcinogens. In contrast, SBTI and pepstatin, which did not inhibit this activity, also do not suppress malignant transformation. These results suggest that this enzyme may be involved in attainment of the transformed state.

Protease inhibitor suppression of colon and anal gland carcinogenesis induced by dimethylhydrazine

In the present study, we examined the ability of chymostatin, a highly specific inhibitor of chymotrypsin, to suppress dimethylhydrazine-induced colon carcinogenesis, and the dose-response relationship for an extract of soybeans containing the Bowman-Birk inhibitor (BBI) to suppress dimethylhydrazine-induced colon carcinogenesis, when added to the diet of mice. Our results showed that: (i) diets containing 0.1% BBI reduced the incidence of adenocarcinomas of the colon approximately 50%, but had no effect on the incidence of squamous cell carcinomas of the anal gland; (ii) the suppressive effect requires protease inhibitor activity, as the autoclaved BBI, in which all protease inhibitory activity has been destroyed, was ineffective at suppressing the incidence of adenocarcinomas; (iii) chymostatin suppressed the incidence of squamous cell carcinomas of the anal gland, but not adenocarcinomas of the colon; and (iv) the growth rates of the animals were the same in each of the experimental groups. Our results indicate that the levels of anticarcinogenic protease inhibitors present in the diets of these animals do not have any adverse effects on the growth or general health of the animals.

Effects of protease inhibitors on c‐myc expression in normal and transformed C3H 10T1/2 cell lines

In the present study, the effect of protease inhibitors on c-myc expression in normal and transformed C3H 10T1/2 cells was examined. Steady-state c-myc RNA levels were reduced in normal proliferating C3H 10T1/2 cells grown in medium containing antipain, leupeptin, and Bowman Birk inhibitor (BBI). These protease inhibitors have been shown previously to suppress transformation yields in carcinogen-exposed cells. A lesser reduction in c-myc RNA levels was observed when cells were grown in the presence of protease inhibitors that do not suppress carcinogenesis and when transformed C3H 10T1/2 cell populations were grown in the presence of the anticarcinogenic protease inhibitors. Studies to determine the effects of antipain on the stability of the c-myc message and on c-myc transcription rates were also performed. The half-life of the c-myc message increased from 10 to 40 min when cells were grown in antipain; cycloheximide further stabilized the c-myc message. Interestingly, nuclear run-off experiments showed that antipain had no effect on c-myc transcription rates. These data suggest that proteases may be involved in the regulation of c-myc RNA expression in normal C3H 10T1/2 cells, possibly by a posttranscriptional mechanism.

A serine protease activity in C3H/10T1/2 cells that is inhibited by anticarcinogenic protease inhibitors.

Several different protease inhibitors have the ability to suppress transformation in vitro and carcinogenesis in vivo. The mechanism(s) by which protease inhibitors suppress carcinogenesis, however, is not fully understood. Presumably, these agents inhibit one or more intracellular proteases whose functions are essential for the induction and/or expression of the transformed phenotype. We have isolated an endopeptidase activity capable of hydrolyzing the substrate Boc-Val-Pro-Arg-MCA (Boc = butoxycarbonyl; MCA = 7-amino-4-methylcoumarin) from C3H/10T1/2 mouse embryo fibroblast cells. This intracellular protease was inhibited by the soybean-derived Bowman-Birk inhibitor (BBI), chymostatin, and L-1-tosylamido-2-phenylethyl chloromethyl ketone, all of which have anticarcinogenic activity, but was unaffected by soybean trypsin inhibitor, which lacks anticarcinogenic activity. Other protease inhibitors affected the proteolytic activity to an extent that correlates with their relative ability to suppress transformation in vitro. The enzyme has a mass of about 70 kDa, contains a single subunit, and exhibits maximal activity at pH 7.0. Diisopropyl fluorophosphate covalently binds to this enzyme and blocks its activity, indicating that the enzyme is a serine protease. We have previously demonstrated that several protease inhibitors are effective suppressors of radiation-induced transformation of C3H/10T1/2 cells. Since these agents reduce the Boc-Val-Pro-Arg-MCA-hydrolyzing activity to an extent that correlates with their ability to inhibit malignant transformation in vitro, this endopeptidase activity may be a cellular target of the anticarcinogenic protease inhibitors.

Inhibition of carcinogen-induced chromosomal aberrations by an anticarcinogenic protease inhibitor.

It was hypothesized that chemicals- and radiation-induced carcinogenesis might require at least two specific chromosomal events that must coincide within a single target cell: (i) induction of chromosomal changes, possibly mutations, that are recessive and therefore latent in diploid somatic cells and (ii) aberrant mitotic segregation events that will convert the heterozygous cell, created by the first process, into a homozygous or hemizygous cell through chromosomal rearrangements. Hence, we tested the prediction that an inhibitor of induced carcinogenesis may inhibit one or both of these chromosomal events by studying the effects of antipain, a protease inhibitor and known inhibitor of carcinogenesis, on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutagenesis, chromosomal aberrations, sister chromatid exchanges, and cell killing in V79 Chinese hamster cells. We show that antipain inhibited MNNG-induced chromosomal exchanges and all other chromosomal aberrations exclusively. This results leads us to postulate that MNNG-induced DNA lesions cause chromosomal aberrations which arise through an antipain-sensitive cellular process, that some chromosomal rearrangement is a rate-limiting step in carcinogenesis, and that mutagenesis alone, if required, is not sufficient to accomplish carcinogenesis.