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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 > or = 80 micrograms/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.

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Bowman-Birk inhibitor (BBI) is a soybean-derived anticarcinogenic protease inhibitor that was shown to potentiate the cytotoxicity of cisplatin in our previous studies. To assess the potential of BBI as a sensitizing agent for the chemotherapy of cisplatin-resistant cancers, we evaluated the effects of a soybean concentrate enriched in BBI (known as BBI concentrate or BBIC) on cell growth and clonogenic survival of a human ovarian cancer cell line, A2780, and its cisplatin-resistant sublines, C30, and C200. The presence of BBI and BBIC in the cell culture, medium reduced the clonogenic survival of the A2780, C30, and C200 cells in a dose-dependent manner and enhanced cisplatin-induced growth inhibition and/or cytotoxicity. BBIC alone showed greater inhibitory effects on growth in the cisplatin-resistant cell lines. These results suggest that BBI and BBIC could be useful agents for the treatment of cancers, especially with cisplatin, in tumors resistant to this important anticancer agent.

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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.

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Bowman-Birk protease inhibitor (BBI) is a potent anticarcinogen that suppresses malignant transformation at nanomolar concentrations. Small amounts of BBI in its native form can be measured by immunoassay using specific monoclonal antibodies (MAbs); however, the MAbs currently available are not capable of detecting BBI metabolites in human body fluids. To develop new reagents for the study of BBI exposure and pharmacokinetics, we produced four MAbs, designated 3B6, 3E3, 4H8 and 5G2, from hybridomas derived from a mouse immunized with reductively modified BBI. The epitopes recognized by the four MAbs were characterized using BBI in its native form or modified by different methods. MAb 3B6 reacted with native BBI. Partial reduction of BBI with 720 Gy of gamma radiation in an oxygen-free solution of 100 mM formate increased the reactivity of BBI with 3B6; however, extensive reduction of BBI with 100 mM DL-dithiothreitol (DTT) completely abolished this antigenic reactivity. In contrast, the other three MAbs reacted with BBI molecules that had been reduced either with 720 Gy of radiation in formate solution or with DTT. Alkylation of the radiochemically reduced BBI with N-ethylmaleimide further increased the reactivity of BBI with 3E3, 4H8 and 5G2, possibly by preventing the formation of new disulfide bonds within the BBI molecules. The binding of 4H8 and 5G2 to BBI antigen was inhibited by the binding of 3E3, and vice versa. Thus, the epitopes recognized by 3E3, 4H8 and 5G2 are probably located close to one another on the reduced BBI molecules. These three MAbs were able to react with BBI metabolites in urine samples collected from volunteers after oral administration of BBI. The ability of these MAbs to detect BBI metabolites indicates that BBI may be reductively modified in vivo and these MAbs may be useful reagents for monitoring the uptake of BBI into human tissues in cancer chemoprevention studies with BBI.

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Rapidly growing Swiss 3T3 fibroblasts possess a bumetanide-sensitive K+ transport system that is dependent on both Na+ and Cl- ions; a smaller bumetanide-insensitive component of K+ transport is also present. In cells brought to the quiescent state by 8-11 days of incubation without a medium change, the bumetanide-sensitive rate of transport was reduced by 63%; the bumetanide-insensitive rate did not change. Removal of dialyzed fetal calf serum from the uptake medium resulted in a substantial reduction in bumetanide-sensitive uptake in both rapidly growing cells (33% reduction) and quiescent cells (68% reduction) but had no effect on bumetanide-insensitive uptake. Insulin was almost as effective as dialyzed fetal calf serum in stimulating bumetanide-sensitive uptake; insulin was maximally stimulatory at 2.5 micrograms/ml. The combination of insulin, epidermal growth factor, and arginine-vasopressin was maximally effective in stimulating both bumetanide-sensitive K+ uptake and 3H-thymidine incorporation in quiescent cells; bumetanide, however, did not interfere with the hormonal stimulation of DNA synthesis. Thus, the bumetanide-sensitive K+ transport system is not necessary for such stimulation to occur. Furthermore, concentrations of hormones which stimulated significant levels of DNA synthesis produced no elevation in the intracellular concentration of K+. We conclude that the bumetanide-sensitive pathway of K+ transport is modulated by serum and by mitogenic hormones, but does not play a role in the stimulation of DNA synthesis by these factors.

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The performance of an automated constant-current coulometric system for the assay of ascorbic acid and sodium ascorbate is described. After loading, it is capable of analyzing 25 samples and printing out the titer values with no operator attention for 2.5 hr. Under optimum conditions, ascertained by evaluating various electrochemical parameters, the accuracy and precision (95% ts) were found to be +/- 0.3%.

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