The effect of concentration and exposure time on soybean protease inhibitor anticancer activity

Soybean protease inhibitors (PI) have been shown to suppress the growth of cancer cells at concentrations below those which produce adverse dietary consequences. However, the combined effects of different PI concentrations and exposure times on cancer growth is not fully understood. The objective of this study was to investigate the effect of different PI concentrations and exposure times on the growth of human cancer cell lines in-vitro. Bowman Birk inhibitor concentrate (BBIC) was prepared from soybean as described by Kassell (1970) and characterized in terms of the chymotrypsin inhibitory activity (CIA) using a standard UV spectrophotometric assay. The crude protein content for BBIC samples was determined by the Ninhydrin method after sample hydrolysis by heating with NaOH (10M) at 121 ^oC for 15 minutes. Anticancer activity was measured using cultured human colon cancer (HT29) or breast cancer (MDA-MB-231) cells pre-grown for 24 hrs using 96-well plates. Cells (1x 10⁴ / well) were exposed to BBIC (5-5000 μg/ ml), dissolved with growth media, for 1 to 4.5 days. Inhibition of cell proliferation was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Soybean BBIC used for this study had 13.2% crude protein and a specific activity of 54,240 CIA / g (protein). BBIC (>50-5000 μg/ ml) produced a maxium of 35% or 55% inhibition of colon and breast cancer cells, respectively. Low levels of BBIC (5-50 μg/ ml) stimulated cell proliferation compared to controls without added inhibitor. There was a significant reduction in the level of inhibition of cancer cells after 4.5 days compared to 1day of exposure. These findings indicate that longer exposure leads to the resistance of colon and breast cancer cells towards soybean BBIC. Further studies are in progress to determine the mechanism of resistance.