Nanomolar concentrations of Bowman-Birk soybean protease inhibitor suppress x-ray-induced transformation in vitro.

Abstract

Experiments reported here indicate a crude soybean extract, if defatted with acetone, effectively blocks cell transformation in vitro. An active component of this crude extract is the Bowman-Birk trypsin and chymotrypsin inhibitor. The chymotrypsin-inhibitory region of the Bowman-Birk inhibitor is responsible for suppressing in vitro transformation. Another low molecular weight soybean trypsin inhibitor does' not significantly suppress transformation. The Bowman-Birk inhibitor (i) has an irreversible effect on the transformation process, (ii) can suppress radiation-induced transformation even when added to cultures many days after the carcinogen exposure, and (iii) is effective in its ability to suppress transformation when present in the medium at a concentration as low as 0.125 nM.We have reported that protease inhibitors, including the Bowman-Birk trypsin and chymotrypsin inhibitor from soybeans, have the ability to suppress x-ray-induced malignant transformation in C3H/10T½ cells (refs. 1-4; reviewed (4,9). We now report that (i) a crude extract of the inhibitor, which can be obtained in reasonable quantities for use in animal carcinogenesis experiments, has the ability to inhibit transformation in vitro; (ii) other soybean protease inhibitors, or other compounds present in our crude extract, lack the ability to suppress transformation in vitro; (iii) the chymotrypsin-inhibitory site of BBI is involved in the suppressive effects of the BBI on transformation; (iv) the lowest effective dose of the protease inhibitor preparations that has the ability to suppress transformation in vitro is 0.125 nM. MATERIALS AND METHODSThe C3H/10T½2 transformation assay was developed by Reznikoff et al. (10,11) and modified for use in radiationinduced transformation experiments (1-5). The details of our experimental techniques for radiation-induced transformation experiments using C3H/10T½/ cells and protease inhibitors have been described (1)(2)(3)(4)(5). Stock cultures were maintained in 60-mm Petri dishes and were passed by subculturing at a 1:20 dilution every 7 days. The cells used were in passages 7-12. They were grown in a humidified 5% CO2 in air atmosphere at 37°C in Eagle's basal medium supplemented with 10% heat-inactivated fetal calf serum and gentamycin. In all transformation experiments, the concentration of serum was reduced to 5% on day 10 and was maintained at this concentration throughout the remainder of the 6-week assay period. Plating efficiencies (PEs) were determined from three plates seeded with a cell density one-fifth of that of the plates used for the transformation assay; these cultures were terminated at 10 days. The various treatment toxicities were considered in the design of the experiments so that all of the dishes used for the transformation assay contained "300 viable cells per dish (initially). Types 2 and 3 foci were scored as transformants; type 3 cells have been found to be tumorigenic in 80-100% of inoculated mice; type 2 cells are tumorigenic in 60-75% of inoculated mice (1-5)...

Anticarcinogenic Factors in Plant Foods: A New Class of Nutrients?

“…They inhibit the transformation of mammalian cells in vitro by radiation and by oncogenes (Kennedy & Little, 1981;Yavelow et al 1983Yavelow et al , 1985Garte er al. 1987) and inhibit the production of oxygen radicals by tumour promoters Troll et al 1987).…”

Detecção de inibidores de proteases em sementes de Punica granatum

“…A metodologia para extração proteica proposta por Yavelow e seus colaboradores 11 foi empregada pela primeira vez em sementes de P. granatum para isolamento e purificação de inibidores de proteases.…”

“…O grão de Glycine max foi adquirido no comércio local de Teófilo Otoni-MG. A extração proteica na G. max foi realizada como medida padrão de comparação para inibidores de proteases, seguindo a metodologia proposta por Yavelow e seus colaboradores, 11 a qual foi adaptada para as sementes de P. granatum.…”

A growth-regulated protease activity that is inhibited by the anticarcinogenic Bowman-Birk protease inhibitor.

“…The anticarcinogenic activity of the soybean-derived Bowman-Birk protease inhibitor (BBI) has been extensively studied in our laboratory (6,8,13,17,(19)(20)(21). The BBI is an 8-kDa protein that effectively inhibits trypsin and chymotrypsin (25) as well as several other proteases (22,(24)(25)(26)(27).…”

“…While protease inhibitors have been shown to have strong anticarcinogenic activity in in vivo and in vitro cancer model systems (for examples, see refs. [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21], relatively little is known about the precise mechanism(s) by which these compounds exert their suppressive effects. We have hypothesized that protease inhibitors block carcinogenesis by inhibiting cellular enzymes involved in induction and/or expression of the transformed phenotype (22)(23)(24).…”