Nutrition and Cancer
ISSN: 0163-5581 (Print) 1532-7914 (Online) Journal homepage: http://www.tandfonline.com/loi/hnuc20
Humoral and cellular immune functions are not compromised by the anticarcinogenic Bowman‐Birk inhibitor Patricia A. Maki & Ann R. Kennedy To cite this article: Patricia A. Maki & Ann R. Kennedy (1992) Humoral and cellular immune functions are not compromised by the anticarcinogenic Bowman‐Birk inhibitor, Nutrition and Cancer, 18:2, 165-173, DOI: 10.1080/01635589209514216 To link to this article: http://dx.doi.org/10.1080/01635589209514216
Published online: 04 Aug 2009.
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Date: 13 November 2015, At: 11:29
Humoral and Cellular Immune Functions Are Not Compromised by the Anticarcinogenic Bowman-Birk Inhibitor Downloaded by [Universite Laval] at 11:29 13 November 2015
Patricia A. Maki and Ann R. Kennedy
Abstract Previous studies have demonstrated that the soybean-derived Bowman-Birk protease inhibitor (BBI) is effective as a cancer chemopreventive agent in several animal model systems. Proteases represent a key component of several aspects of immune function; therefore the immune system is a primary target for potential toxicity. The present investigation examines the effect of dietary and intraperitoneally administered BBI on antibody response to keyhole limpet hemocyanin and delayed-type hypersensitivity response to dinitrochlorobenzene. Primary antibody response was not altered by BBI treatment; however, an elevated secondary response was observed in animals receiving dietary BBI at two weeks of age. This effect was not observed at later time points. No change in delayed-type hypersensitivity response was observed in any of the treatment groups. (Nutr Cancer 18, 165-173, 1992)
Introduction
Recently, there has been increasing interest in the study of cancer chemoprevention. Many food constituents have been shown to have anticarcinogenic properties (1); among them, vitamin A, selenium, and a-tocopherol have shown some promise as potential cancer chemopreventive agents. Protease inhibitors represent one of the most promising classes of agents for the prevention of cancer (2-5). Large amounts of protease inhibitors are consumed by cultures whose diets are rich in vegetables and legumes (6). Epidemiological data suggest that populations having a high intake of dietary protease inhibitors have a lower-than-normal incidence of breast, colon, and prostate cancer (7,8). Considerable experimental evidence is accumulating to support the role of protease inhibitors as anticarcinogens. Several protease inhibitors have been shown to suppress oncogenic transformation in vitro induced by both chemical and physical carcinogens (reviewed in Ref. 9). Protease inhibitors have also been shown to suppress a number of other The authors are affiliated with the Department of Radiation Oncology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.
Copyright © 1992, Lawrence Erlbaum Associates, Inc.
Downloaded by [Universite Laval] at 11:29 13 November 2015
in vitro phenomena that are associated with malignant transformation, such as c-myc expression (10,11), c-fos expression (12), ras-induced transformation (13), and chromosome aberrations in cells from patients with Bloom's syndrome; such phenomena are thought to predispose these patients to a higher-than-normal incidence of cancer (14). Protease inhibitors have also been shown to be highly effective in suppressing carcinogenesis in vivo (2,3,5). One of the most promising of the anticarcinogenic protease inhibitors is the soybeanderived Bowman-Birk inhibitor (BBI). Soybean-based diets, which are rich in protease inhibitors, have been shown to reduce breast cancer in irradiated rats (15) and spontaneous hepatocarcinogenesis in C3H/HeN mice (16). Studies from this laboratory have demonstrated that BBI can suppress dimethylhydrazine- (DMH) induced colon carcinogenesis in mice (17-19), DMH-induced angiosarcomas and nodular hyperplasia of the liver (19), dimethylbenz[ar]anthracene-induced cheek pouch carcinogenesis in hamsters (20; unpublished data), 3-methylcholanthrene-induced lung tumor development in mice (21), and N-nitrosobenzylamine-induced esophageal cancer in mice (von Hofe, Newberne, and Kennedy, unpublished observations). In light of these promising reports, questions have been raised regarding the potential toxicity resulting from chronic administration of protease inhibitors as cancer chemopreventive agents. Primary among these concerns is the potential for these protease inhibitors to suppress immune function, thus compromising the animals' ability to mount a response to environmental pathogens. Proteases have been shown to be an integral part of several aspects of immune function, such as antigen processing (22) and the cytolytic mechanisms of cytotoxic T, natural killer (NK), and lymphokine-activated killer (LAK) cells (23-27). It was therefore necessary to examine the effect of BBI on both humoral and cellular immune function. Materials and Methods
Alkaline phosphatase substrate, incomplete Fruends adjuvant, l-chloro-2,4-dinitrochlorobenzene (DNCB), and alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G (IgG) were obtained from Sigma Chemical (St. Louis, MO). Keyhole limpet hemocyanin (KLH) was obtained from Calbiochem (San Diego, CA). Borate-buffered saline (BBS) consisted of 0.1 M boric acid, 0.025 M borax, and 0.075 M sodium chloride (pH 8.2). Sodium carbonate substrate buffer was comprised of 0.05 M sodium carbonate and 1.0 mM MgCl2 (pH 9.8). Animals Male CD-I mice, five to six weeks of age (Charles River Breeding Laboratories, Wilmington, MA) were housed 10 per cage and maintained on standard rodent chow until assigned to treatment groups. Throughout the experiment, animals were maintained in climate-controlled rooms with a 12:12-hour light-dark cycle. At seven to eight weeks of age, animals were randomly assigned to groups and treatments were initiated. Diets In experiments to examine the effect of dietary BBI on immune function, all treatment groups were fed a standard diet [AIN-76A, American Institute of Nutrition purified diet for rats and mice (28)] prepared by Zeigler Brothers (Gardner, PA). In some treatment groups, the standard diet was modified by the addition of BBI prepared as previously described (29). Briefly, acetone-defatted soybean flour was extracted with 60% ethanol, and the material in the acidified extract was resuspended in water, dialyzed, and lyophilized. The resulting
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freeze-dried crude extract, termed BBI concentrate (BBIC), contains 50% protease inhibitors by weight, of which BBI represents approximately 10%. The resulting BBIC preparation is then purified by DEAE-cellulose chromatography to produce purified BBI (pBBI). Autoclaved BBIC (aBBIC) was prepared by autoclaving BBIC for 90 minutes. The resulting preparation was assayed for ability to inhibit trypsin and chymotrypsin activity to verify that protease inhibitory activity had been completely eliminated. The mice were assigned to treatment groups receiving standard diet, standard diet supplemented with 0.1% pBBI, or standard diet supplemented with 0.5% BBIC or aBBIC. Additional groups of mice were treated with 2 mg of pBBI (in 0.2 ml of sterile 0.9% saline) or an equivalent volume of saline.
Downloaded by [Universite Laval] at 11:29 13 November 2015
Antibody Titer to KLH At 2,4, 8, and 16 weeks after initiation of BBI-containing diets, five animals per treatment group were immunized with 100 pg of KLH (0.1 ml of a 1-mg/ml solution in phosphatebuffered saline). Nine to ten days after immunization, blood samples were taken from the tail. Serum was isolated by centrifugation at 16,000 g for 10 minutes. Antibody titers to KLH were then determined by enzyme-linked immunosorbent assay (ELISA). ELISA Ninety-six-well microtiter plates were coated with KLH by adding 50 p\ of a 20-/tg/ml (in BBS) solution to each test well and incubating overnight at 4°C. The plates are then incubated with 200 fil of 1% bovine serum albumin for one hour at room temperature. Antiserum from treated mice was serially diluted and added to the KLH-coated plates, which were washed three times with BBS. The plates are incubated for four to six hours at room temperature and washed three times, and goat anti-mouse IgG (diluted 1:500 with 1% bovine serum albumin in BBS) is added to each test well. The plates are incubated overnight at 4°C. Alkaline phosphate substrate (1 mg/ml in NaCO3 buffer) is added to test and blank wells. The colored reaction product is allowed to develop for 30 minutes and then quantified by a Microtiter plate reader. Antibody titers are calculated from the plot of absorbance versus serum dilution. Antibody titer is defined as the reciprocal of the serum dilution at half-maximal absorbance. Delayed-Type Hypersensitivity Assay Animals maintained on BBI-containing diets or treated intraperitoneally with pBBI three times per week (2 mg in 0.1 ml of normal saline) were immunized with 100 /*g of DNCB (in acetone-olive oil, 1:4) intraperitoneally 2, 4, 8, and 16 weeks after initiation of diets. Nine days later, each mouse was challenged in the left hind footpad with 30 fil of DNCB. The right foot was used for control and was injected with an equal volume of vehicle. Twenty-four hours later, the footpad swelling increment (left foot minus right foot) was determined by use of an engineer's caliper. Triplicate measurements were taken on each foot. The results are expressed as means ± SE. Statistical analysis was by analysis of variance for differences between groups. Results To determine whether dietary BBI would compromise the ability to produce antibodies in response to foreign antigen, animals were fed BBI-containing diets beginning at seven to eight weeks of age and were immunized with KLH at various time points. Nine to ten days after immunization, blood samples were taken from the tail and the serum was analyzed by ELISA for antibodies to KLH.
Vol. 18, No. 2
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No difference in the ability to produce anti-KLH antibodies was observed after primary immunization in animals that were immunized two weeks after the initiation of BBIcontaining diets (Figure 1A). When these animals were given a secondary antigen challenge (100 /ig of KLH ip 21 days after primary immunization), higher antibody production was observed in animals maintained on the pBBI- and BBIC-containing diet (Figure IB). It has been demonstrated that the ability of agents to alter immune function can be dependent on the route of administration (30). We therefore wanted to determine whether the ability of animals to respond to foreign antigen was compromised when BBI was administered by intraperitoneal injection. Animals were injected intraperitoneally with 2 mg of pBBI or 0.9% saline three times per week. Animals were immunized with KLH two weeks after the first BBI injection, and a secondary immunization was given 21 days later. Serum samples were analyzed for anti-KLH antibodies 9-10 days after immunization. No difference in antibody production was observed between pBBI- or saline-treated animals after either primary or secondary immunization (Figure 2). Treatment was continued and antibody titers were determined at various time intervals. No difference in antibody response was observed at any of the time points tested (Table 1). Inasmuch as antibody production is a measurement of only one arm of the immune
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ISSN: 0163-5581 (Print) 1532-7914 (Online) Journal homepage: http://www.tandfonline.com/loi/hnuc20
Humoral and cellular immune functions are not compromised by the anticarcinogenic Bowman‐Birk inhibitor Patricia A. Maki & Ann R. Kennedy To cite this article: Patricia A. Maki & Ann R. Kennedy (1992) Humoral and cellular immune functions are not compromised by the anticarcinogenic Bowman‐Birk inhibitor, Nutrition and Cancer, 18:2, 165-173, DOI: 10.1080/01635589209514216 To link to this article: http://dx.doi.org/10.1080/01635589209514216
Published online: 04 Aug 2009.
Submit your article to this journal
Article views: 6
View related articles
Citing articles: 4 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=hnuc20 Download by: [Universite Laval]
Date: 13 November 2015, At: 11:29
Humoral and Cellular Immune Functions Are Not Compromised by the Anticarcinogenic Bowman-Birk Inhibitor Downloaded by [Universite Laval] at 11:29 13 November 2015
Patricia A. Maki and Ann R. Kennedy
Abstract Previous studies have demonstrated that the soybean-derived Bowman-Birk protease inhibitor (BBI) is effective as a cancer chemopreventive agent in several animal model systems. Proteases represent a key component of several aspects of immune function; therefore the immune system is a primary target for potential toxicity. The present investigation examines the effect of dietary and intraperitoneally administered BBI on antibody response to keyhole limpet hemocyanin and delayed-type hypersensitivity response to dinitrochlorobenzene. Primary antibody response was not altered by BBI treatment; however, an elevated secondary response was observed in animals receiving dietary BBI at two weeks of age. This effect was not observed at later time points. No change in delayed-type hypersensitivity response was observed in any of the treatment groups. (Nutr Cancer 18, 165-173, 1992)
Introduction
Recently, there has been increasing interest in the study of cancer chemoprevention. Many food constituents have been shown to have anticarcinogenic properties (1); among them, vitamin A, selenium, and a-tocopherol have shown some promise as potential cancer chemopreventive agents. Protease inhibitors represent one of the most promising classes of agents for the prevention of cancer (2-5). Large amounts of protease inhibitors are consumed by cultures whose diets are rich in vegetables and legumes (6). Epidemiological data suggest that populations having a high intake of dietary protease inhibitors have a lower-than-normal incidence of breast, colon, and prostate cancer (7,8). Considerable experimental evidence is accumulating to support the role of protease inhibitors as anticarcinogens. Several protease inhibitors have been shown to suppress oncogenic transformation in vitro induced by both chemical and physical carcinogens (reviewed in Ref. 9). Protease inhibitors have also been shown to suppress a number of other The authors are affiliated with the Department of Radiation Oncology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104.
Copyright © 1992, Lawrence Erlbaum Associates, Inc.
Downloaded by [Universite Laval] at 11:29 13 November 2015
in vitro phenomena that are associated with malignant transformation, such as c-myc expression (10,11), c-fos expression (12), ras-induced transformation (13), and chromosome aberrations in cells from patients with Bloom's syndrome; such phenomena are thought to predispose these patients to a higher-than-normal incidence of cancer (14). Protease inhibitors have also been shown to be highly effective in suppressing carcinogenesis in vivo (2,3,5). One of the most promising of the anticarcinogenic protease inhibitors is the soybeanderived Bowman-Birk inhibitor (BBI). Soybean-based diets, which are rich in protease inhibitors, have been shown to reduce breast cancer in irradiated rats (15) and spontaneous hepatocarcinogenesis in C3H/HeN mice (16). Studies from this laboratory have demonstrated that BBI can suppress dimethylhydrazine- (DMH) induced colon carcinogenesis in mice (17-19), DMH-induced angiosarcomas and nodular hyperplasia of the liver (19), dimethylbenz[ar]anthracene-induced cheek pouch carcinogenesis in hamsters (20; unpublished data), 3-methylcholanthrene-induced lung tumor development in mice (21), and N-nitrosobenzylamine-induced esophageal cancer in mice (von Hofe, Newberne, and Kennedy, unpublished observations). In light of these promising reports, questions have been raised regarding the potential toxicity resulting from chronic administration of protease inhibitors as cancer chemopreventive agents. Primary among these concerns is the potential for these protease inhibitors to suppress immune function, thus compromising the animals' ability to mount a response to environmental pathogens. Proteases have been shown to be an integral part of several aspects of immune function, such as antigen processing (22) and the cytolytic mechanisms of cytotoxic T, natural killer (NK), and lymphokine-activated killer (LAK) cells (23-27). It was therefore necessary to examine the effect of BBI on both humoral and cellular immune function. Materials and Methods
Alkaline phosphatase substrate, incomplete Fruends adjuvant, l-chloro-2,4-dinitrochlorobenzene (DNCB), and alkaline phosphatase-conjugated goat anti-mouse immunoglobulin G (IgG) were obtained from Sigma Chemical (St. Louis, MO). Keyhole limpet hemocyanin (KLH) was obtained from Calbiochem (San Diego, CA). Borate-buffered saline (BBS) consisted of 0.1 M boric acid, 0.025 M borax, and 0.075 M sodium chloride (pH 8.2). Sodium carbonate substrate buffer was comprised of 0.05 M sodium carbonate and 1.0 mM MgCl2 (pH 9.8). Animals Male CD-I mice, five to six weeks of age (Charles River Breeding Laboratories, Wilmington, MA) were housed 10 per cage and maintained on standard rodent chow until assigned to treatment groups. Throughout the experiment, animals were maintained in climate-controlled rooms with a 12:12-hour light-dark cycle. At seven to eight weeks of age, animals were randomly assigned to groups and treatments were initiated. Diets In experiments to examine the effect of dietary BBI on immune function, all treatment groups were fed a standard diet [AIN-76A, American Institute of Nutrition purified diet for rats and mice (28)] prepared by Zeigler Brothers (Gardner, PA). In some treatment groups, the standard diet was modified by the addition of BBI prepared as previously described (29). Briefly, acetone-defatted soybean flour was extracted with 60% ethanol, and the material in the acidified extract was resuspended in water, dialyzed, and lyophilized. The resulting
166
Nutrition and Cancer 1992
freeze-dried crude extract, termed BBI concentrate (BBIC), contains 50% protease inhibitors by weight, of which BBI represents approximately 10%. The resulting BBIC preparation is then purified by DEAE-cellulose chromatography to produce purified BBI (pBBI). Autoclaved BBIC (aBBIC) was prepared by autoclaving BBIC for 90 minutes. The resulting preparation was assayed for ability to inhibit trypsin and chymotrypsin activity to verify that protease inhibitory activity had been completely eliminated. The mice were assigned to treatment groups receiving standard diet, standard diet supplemented with 0.1% pBBI, or standard diet supplemented with 0.5% BBIC or aBBIC. Additional groups of mice were treated with 2 mg of pBBI (in 0.2 ml of sterile 0.9% saline) or an equivalent volume of saline.
Downloaded by [Universite Laval] at 11:29 13 November 2015
Antibody Titer to KLH At 2,4, 8, and 16 weeks after initiation of BBI-containing diets, five animals per treatment group were immunized with 100 pg of KLH (0.1 ml of a 1-mg/ml solution in phosphatebuffered saline). Nine to ten days after immunization, blood samples were taken from the tail. Serum was isolated by centrifugation at 16,000 g for 10 minutes. Antibody titers to KLH were then determined by enzyme-linked immunosorbent assay (ELISA). ELISA Ninety-six-well microtiter plates were coated with KLH by adding 50 p\ of a 20-/tg/ml (in BBS) solution to each test well and incubating overnight at 4°C. The plates are then incubated with 200 fil of 1% bovine serum albumin for one hour at room temperature. Antiserum from treated mice was serially diluted and added to the KLH-coated plates, which were washed three times with BBS. The plates are incubated for four to six hours at room temperature and washed three times, and goat anti-mouse IgG (diluted 1:500 with 1% bovine serum albumin in BBS) is added to each test well. The plates are incubated overnight at 4°C. Alkaline phosphate substrate (1 mg/ml in NaCO3 buffer) is added to test and blank wells. The colored reaction product is allowed to develop for 30 minutes and then quantified by a Microtiter plate reader. Antibody titers are calculated from the plot of absorbance versus serum dilution. Antibody titer is defined as the reciprocal of the serum dilution at half-maximal absorbance. Delayed-Type Hypersensitivity Assay Animals maintained on BBI-containing diets or treated intraperitoneally with pBBI three times per week (2 mg in 0.1 ml of normal saline) were immunized with 100 /*g of DNCB (in acetone-olive oil, 1:4) intraperitoneally 2, 4, 8, and 16 weeks after initiation of diets. Nine days later, each mouse was challenged in the left hind footpad with 30 fil of DNCB. The right foot was used for control and was injected with an equal volume of vehicle. Twenty-four hours later, the footpad swelling increment (left foot minus right foot) was determined by use of an engineer's caliper. Triplicate measurements were taken on each foot. The results are expressed as means ± SE. Statistical analysis was by analysis of variance for differences between groups. Results To determine whether dietary BBI would compromise the ability to produce antibodies in response to foreign antigen, animals were fed BBI-containing diets beginning at seven to eight weeks of age and were immunized with KLH at various time points. Nine to ten days after immunization, blood samples were taken from the tail and the serum was analyzed by ELISA for antibodies to KLH.
Vol. 18, No. 2
167
Downloaded by [Universite Laval] at 11:29 13 November 2015
No difference in the ability to produce anti-KLH antibodies was observed after primary immunization in animals that were immunized two weeks after the initiation of BBIcontaining diets (Figure 1A). When these animals were given a secondary antigen challenge (100 /ig of KLH ip 21 days after primary immunization), higher antibody production was observed in animals maintained on the pBBI- and BBIC-containing diet (Figure IB). It has been demonstrated that the ability of agents to alter immune function can be dependent on the route of administration (30). We therefore wanted to determine whether the ability of animals to respond to foreign antigen was compromised when BBI was administered by intraperitoneal injection. Animals were injected intraperitoneally with 2 mg of pBBI or 0.9% saline three times per week. Animals were immunized with KLH two weeks after the first BBI injection, and a secondary immunization was given 21 days later. Serum samples were analyzed for anti-KLH antibodies 9-10 days after immunization. No difference in antibody production was observed between pBBI- or saline-treated animals after either primary or secondary immunization (Figure 2). Treatment was continued and antibody titers were determined at various time intervals. No difference in antibody response was observed at any of the time points tested (Table 1). Inasmuch as antibody production is a measurement of only one arm of the immune
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