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Nutrition and Disease

Mechanisms Associated with Dose-Dependent Inhibition of Rat Mammary Carcinogenesis by Dry Bean (Phaseolus vulgaris, L.)^1–3,

⁴ Cancer Prevention Laboratory, Department of Horticulture and ⁵ Department of Soil and Crop Sciences, Colorado State University, Fort Collins, CO 80523 and ⁶ Department of Preventive Medicine and Biometrics, University of Colorado Health Sciences Center, Denver, CO 80262

^* To whom correspondence should be addressed. E-mail: henry.thompson{at}colostate.edu.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
The purpose of this study was to determine whether a dry bean (Phaseolus vulgaris, L.) containing diet exerts an inhibitory effect on mammary carcinogenesis in a well-characterized rodent model for breast cancer. Twenty-one-d-old female Sprague Dawley rats were given an intraperitoneal injection of 1-methyl-1-nitrosourea and 7 d after carcinogen injection were randomized to 1 of 5 groups fed a modification of the AIN-93G diet formulation containing 0, 7.5, 15, 30, or 60% (wt:wt) small red dry bean incorporated as cooked, freeze-dried, and milled powder. All experimental diets had the same macronutrient content based on proximate analysis. Compared with the control group, dry bean consumption resulted in dose-dependent reductions in mammary cancer incidence (P = 0.046), cancer multiplicity (P = 0.001), and tumor burden (P = 0.01). Dry bean consumption was associated with dose-dependent reductions in plasma concentrations of glucose, insulin, insulin-like growth factor-1, C-reactive protein, and interleukin-6 in food-deprived rats. Analysis of mammary adenocarcinomas indicated that a dominant mechanism accounting for reduced tumor burden was the induction of apoptosis. B cell lymphoma 2 and X-linked inhibitor of apoptosis protein levels decreased and BCL-2–associated X protein increased with increasing dry bean consumption, findings consistent with the induction of apoptosis via the mitochondrial pathway. These data demonstrate that a legume without noteworthy content of isoflavones inhibits the development of mammary carcinogenesis and are consistent with a recent report from the Nurses Health Study that bean or lentil intake is associated with a lower risk for breast cancer.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

The legume family, Fabaceae, includes all plants that produce seeds within a pod. Pulses are defined by the FAO as annual leguminous crops yielding from 1 to 12 grains or seeds of variable size, shape, and color within a pod that are used for food and animal feed. The term pulses, as used by the FAO, is reserved for crops harvested solely for their dry grain. This therefore excludes green beans and green peas, which are considered vegetable crops. Also excluded are crops that are mainly grown for oil extraction, such as soybean [Glycine max (L) Merr.] or peanut (Arachis hypogeal L.), and crops that are used exclusively for animal forages, such as clovers (Trifolium sp.) or alfalfa (Medicago sativa L.). Of the legumes investigated for their effects on the development of breast cancer, soybeans and the isoflavone-rich foods derived from soy have been reported to have protective effects against experimentally induced breast cancer (2–4). However, there is no information regarding whether pulses such as dry bean (Phaseolus vulgaris, L.), which are 2 to 3 orders of magnitude lower in isoflavone content than soy meal, affect experimentally induced breast cancer (5).

When eaten as a staple food, dry beans are consumed daily and in large quantities; thus they could represent an excellent vehicle with which to deliver health-promoting chemicals to the population. However, although pulse consumption is high in certain regions of Africa, Asia, and Central and South America, consumption of pulses such as dry bean in the United States is quite low (7.5 g/d) (6). An increase in dry bean consumption may have important impact if the results from the Nurses Health Study are shown to have a biological basis (1).

The current study was conducted to determine the ability of cooked, commercially processed dry bean to inhibit breast cancer using a well-characterized rodent model at doses of dry bean achievable in the human diet and at higher doses reported to reduce the occurrence of experimentally induced colon cancer. Because mammary carcinogenesis was inhibited, additional experiments were conducted to identify candidate mechanisms. Those experiments included the evaluation of plasma for growth factors and cytokines that have been implicated as playing causal roles in carcinogenesis and the investigation of cellular and molecular processes possibly underlying the effects of dry bean intake on tumor burden.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Chemicals. Primary antibodies used in this study were anti-cyclin D1, anti-E2F transcription factor 1 (E2F1)⁷, and anti-p27^Kip1 from Thermo Fisher Scientific; anti-retinoblastoma (Rb), anti-B cell lymphoma 2 (BCL-2), anti-X-linked inhibitor of apoptosis protein (XIAP), and anti-BCL-2–associated X protein (BAX) from BD Biosciences; anti-apoptosis protease-activating factor 1 (APAF1) from Millipore; and anti-rabbit immunoglobulin horseradish peroxidase (HRP) conjugated secondary antibody, as well as LumiGLO reagent with peroxide, all from Cell Signaling Technology. Anti-p21^Cip1 and anti-mouse Ig HRP-conjugated secondary antibody were from Santa Cruz; mouse anti-β-actin primary antibody was obtained from Sigma Chemical; rabbit anti-Ki-67, clone SP6, was obtained from Labvision. Biotinylated donkey anti-rabbit, donkey anti-goat secondary antibodies, and normal donkey serum were obtained from Jackson Immuno Research; HRP-conjugated streptavidin was obtained from Dako; and Stable DAB was obtained from Invitrogen.

    Rats and experimental design. One hundred fifty female Sprague Dawley rats were obtained from Taconic Farms at 20 d of age. Animal rooms were maintained at 22 ± 2°C with 50% relative humidity and a 12-h-light/12-h-dark cycle. During the experiment, rats were weighed 3 times per week. At 21 d of age, rats were injected with 1-methyl-1-nitrosourea (50 mg/kg body weight, intraperitoneal), as previously described (7). For the first week of the study, rats were housed 3 per cage in solid bottomed polycarbonate cages equipped with a food cup; they were given free access to AIN-93G control diet (8). Seven d following carcinogen injection, all rats were randomized into 1 of 5 diet groups based on body weight: 0 (control), 7.5, 15, 30, or 60% (wt:wt) small red bean. Rats consumed the assigned diets ad libitum until the end of the study at 46-d post carcinogen. The postinitiation design of this experiment simulates the promotion/progression events of the disease process, which is highly relevant to women at increased risk for breast cancer and to breast cancer survivors. The work followed ethical guidelines approved by the Colorado State University Animal Care and Use Committee.

    Composition of diets. Dry beans were kindly provided by Archer Daniels Midland as seed from the market class small red dry bean. We used small red bean because it was recently reported that it had the highest oxygen radical absorbance capacity based on comparing serving sizes of over 100 foods that were assessed in the assay (9). Seed was commercially grown at multiple locations and mixed and is therefore representative of the variation in environmental and genetic differences that would typify beans produced and consumed in the United States. Dry bean seed was sent to Bush Brothers & Company for canning and all material was processed according to industry standard methods. Cooked beans were packed in standard brine without the incorporation of any additives. Beans were then sent to Van Drunen Farms where the beans were removed from the cans, drained, and then immediately freeze-dried. The freeze-dried product was milled into a homogeneous powder and sent to Colorado State University where bean powders were stored at –20°C until incorporation into diets. Diets were formulated using specific guidelines (8) and adjusted using data from proximate analysis (Warren Analytical). The diets were formulated to match macronutrient levels (i.e. protein, carbohydrate, and crude fiber) across the diet groups. The differences in macronutrient composition were balanced with purified diet components. The percentage of bean incorporated into the diets is expressed as mass of bean powder in g/100 g of total diet. Control diets consisted of 7.5% crude fiber to correspond to the fiber content of the bean diets. Diet formulations are described (Table 1). Diets were stored at –20°C until fed to the rats. Calculations to determine equivalence to a human diet were based on a 7534-kJ/d human diet consisting of 46% carbohydrate, 34% protein, and 20% fat and a mean water content of 81%. The estimated mass (g) of cooked and canned beans was 256 g/cup with water content estimated at 75–80%. Calculations were conducted with data from ProNutra software (version 3.1, Viocare Technologies). Dietary equivalents (Table 1) were based on these assumptions.

    Plasma analytes. Glucose was measured using a kit obtained from Thermo Fisher Scientific. Insulin was measured by commercial ELISA kit from Millipore. Insulin growth factor-1 (IGF-1) was measured using a commercial rat enzyme immunoassay kit from Diagnostic Systems Laboratories. Interleukin-6 (IL-6) and C-reactive protein (CRP) were measured using ELISA kits from BD Biosciences. All experiments were performed according to the manufacturer's directions included in each kit and analyzed using a SpectraMax M5 Microplate Reader from Molecular Devices.

    Cell proliferation and apoptosis. Ki-67 immunohistochemical staining was used as an index of tumor growth fraction and was determined as previously described (11). Ki-67–stained sections were analyzed using a CAS-200 image analysis system (Bacus Labs). Apoptosis was quantified using the criteria developed by Kerr for its detection (12,13); images of corresponding H&E-stained serial sections were acquired using a Zeiss Axioskop II (Carl Zeiss) at a magnification of 400x. Apoptotic and normal cells were marked and counted using the manual tag tools in Image Pro Plus 4.5 (Media Cybernetics).

    Western blotting. Mammary AC (7–9/group) were analyzed by Western blotting as previously described (14). Protein was visualized using the LumiGLO reagent Western blotting detection system. The chemiluminescence signal was captured using a ChemiDoc (Bio-Rad) that was equipped with a CCD camera having a resolution of 1300 x 1030. Quantity One software (Bio-Rad) was used in the analysis. The actin-normalized scanning density data are reported.

    Statistical analyses. Cancer incidence, cancer multiplicity, cancer latency, and tumor burden were evaluated by logistic regression analysis, Poisson regression analysis, a log rank test, and ANOVA following rank transformation, respectively (15,16). Differences among groups in final body weight, plasma concentrations, and rates of cell proliferation and apoptosis were analyzed by ANOVA with post hoc comparisons by the method of Tukey (17). For Western blots, the actin-normalized scanning density or ratio data were rank transformed and subjected to multivariate ANOVA (18). Data were evaluated using SAS v. 9.1.3, STATA (Stata), or Systat v. 12.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Dry bean consumption was well tolerated by the rats with no grossly apparent evidence of adverse effects during daily evaluations. Dry bean–fed rats grew in comparison to the control group although rats fed 60% (wt:wt) dry bean had a final body weight that was 8.5% lower than the control (Table 2; P < 0.01).

View larger version (13K): [in this window] [in a new window]   FIGURE 1  Effect of dry bean diets on cancer multiplicity (mean number of cancers per rat) and tumor burden (mean tumor mass, g/rat). Values are means ± SEM of the original data, n = 29 or 30. *Different from 0% bean, P < 0.05.

View larger version (42K): [in this window] [in a new window]   FIGURE 2  Effect of dry bean diets on cell proliferation of mammary AC. (A) Immunohistochemical demonstration of Ki-67 in mammary AC showing variable expression with clusters of intensely stained epithelial nuclei (arrows) and an adjacent normal mammary duct with a single positive nucleus (arrowhead). Bar = 10 µm. (B) Values are means ± SEM, n = 7–9. (C) Representative western blot images for each of the cell cycle regulators.

View larger version (45K): [in this window] [in a new window]   FIGURE 3  Effect of dry bean diets on cell apoptosis of mammary AC. (A) H&E-stained mammary AC with numerous apoptotic cells (arrows). Bar = 10 µm. (B) Values are means ± SEM, n = 10. *Different from 0% red bean, P < 0.05. (C) Representative western blot images for each of the apoptosis regulators.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Inhibition of mammary carcinogenesis by dry bean fed in a form typically consumed by human populations is consistent with a recent prospective study that showed that breast cancer risk in the Nurses Health Study II cohort was reduced by 24% in women consuming <1 serving of beans or lentils per month compared with women consuming 2–4 servings per week (1), an effect size similar to that reported at the lowest level of dry bean consumption (Table 2). The cancer inhibitory findings of our study are also consistent with the results from the Polyp Prevention Trial in which it was shown that dry bean consumption is associated with reduced risk for advanced colon cancer. The intervention group with the highest quartile of change in dry bean consumption (41.5 g/d) compared with the lowest quartile of change (5.7 g/d) reduced recurrence of advanced adenomas by 65% (OR = 0.35; 95% CI, 0.18–0.69; P-trend = 0.001) (23). Moreover, animal studies have also provided evidence consistent with an inhibitory effect of dietary dry bean on colon carcinogenesis induced in rats by azoxymethane (24,25), although the doses studied were >59% (wt:wt) in the diet.

    Mechanistic experiments. To explore the chemical basis for breast cancer inhibitory activity, an understanding of what distinguishes dry beans from other pulses and other staple foods is required. Dry bean, when consumed as a food, contains many compounds with potential cancer-preventive activity (26,27). To identify the classes of compounds as well as specific chemicals that account for biological activity of a food containing natural products, such as dry bean, it is typical to conduct bioactivity guided fractionations to facilitate compound isolation and identification. The most common system used for this purpose in cancer research is the growth inhibition of cancer cells in monolayer culture. However, our initial efforts to screen various chemical extracts (e.g. ethanol or acetone extracts) of small red bean for growth inhibitory activity using the human breast cancer cell lines MCF-7 and MDA-MB-468 were negative (data not shown). This suggested to us that dry bean consumption might be mediating effects via perturbing systemic circulating factors, as would be inferred from investigations of the effects of dry bean consumption on type-2 diabetes and heart disease (19,20). For this reason, experiments were conducted to determine the effects of dry bean consumption on plasma indicators of glucose homeostasis and chronic inflammation, both of which have been associated with breast cancer risk (28,29). Dry bean intake reduced circulating levels of glucose and the concentration of 2 proteins, insulin and IGF-1 (Table 3), that are known to affect the development of breast cancer (28,30,31). These effects were achieved even at the lowest levels of dry bean intake. The effects on CRP and IL-6 were less pronounced but still may point to intracellular signaling pathways involved in inflammation and carcinogenesis that are targeted when dry bean consumption is increased (29). Given that growth factors such as IGF-1 and cytokines such as IL-6 have been reported to affect rates of cell proliferation and apoptosis in breast cancer cells (28,29), the effects of dry bean consumption on these cellular processes was further investigated.

The expression of the human Ki-67 protein is strictly associated with cell proliferation, being present during all active phases of the cell cycle (G1, S, G2, and mitosis) but absent from resting cells [G(0)]. This makes Ki-67 an excellent marker for determining growth fraction, i.e. the proportion of cells not in G(0), for a given cell population (32). There was no evidence to indicate that dry bean consumption affected growth fraction in the mammary AC assessed (Fig. 2). However, when lysates of mammary AC were used to determine the ratio of ppRb:pRb, the ratio decreased with increasing dry bean intake and the E2F1 concentration was reduced. This implies that dry bean suppressed the rate of passage of cells from G(1) to S even though the growth fraction was not affected. The fact that levels of E2F1 were reduced is consistent with the Rb data in that E2F1 is normally bound to pRb and is released when Rb is progressively phosphorylated by cyclin-dependent kinases 2 and 4. Free E2F1 is a critical transcriptional factor that promotes the expression of genes involved in the G1/S transition and the synthesis of DNA (33). The data obtained also indicate that effects of dry bean consumption are likely due to the modulation of the activity of cyclin-dependent kinase inhibitors such as p21 rather than to direct effects on the level of cyclin D1, which is generally overexpressed in this model system as well as in the human disease. The effects of dry bean in suppressing the rate of passage of cells from G1 to S are consistent with the reduction in plasma IGF-1 and insulin that affect cell cycle regulation via the mammalian target of rapamycin (34) as well as with the reduced plasma concentrations of IL-6 that affect cell proliferation via glycoprotein 130 (gp130) mediated activation of signaling pathways, of which JAK/STAT and MAP kinase are components and reviewed in (29).

The rate of apoptosis was measured histologically using the criteria initially used by Kerr et al. (12). Recognizing that apoptosis is a very rapid process and that small differences in apoptotic rate can have a major impact on tumor growth, the significant effect of dry bean intake on the rate of apoptosis, a 2-fold increase across the dose response (Fig. 3B), provided strong evidence that dry bean intake was regulating this process. Consequently, the basis for apoptosis induction was evaluated in greater detail. Investigation of BCL-2 family members indicated that at least a component of the proapoptotic effect of dry bean consumption was being mediated by the intrinsic or mitochondrial pathway of cell death induction (Table 4). The mitochondrial pathway has been found to be sensitive to decreases in growth factors such as IGF-1, because IGF-1 regulates tissue levels of activated protein kinase B, which is a survival factor that blocks the occurrence of apoptosis (35). The strong dose-dependent increase in the ratio of BAX:BCL-2 (Table 4) provides an additional indication that the intracellular environment of mammary AC in dry bean-fed rats was highly proapoptotic, an effect consistent with the reduced tumor mass with increasing bean consumption (Table 2). Additional work is necessary to determine whether the receptor-mediated pathway of cell death induction might also be involved, because cytokines are known to act via the extrinsic, receptor-mediated pathway (36).

Overall mean dry bean per capita consumption for the US was estimated at 6 lb/y (2.7 kg/y) (6), 7.5 g/d. These numbers fall well below recommended levels of dry bean consumption, which is 70 g/d (3 cups/wk) based on the current USDA Food Guide Pyramid (37). The current findings provide evidence for health-promoting qualities of dry beans that extend beyond heart disease and type 2 diabetes to cancer. Given the relatively low daily intake of dry beans in the United States, the dose response data reported herein indicate that increased incorporation of dry beans into the diet represents an unappreciated opportunity to reduce the risk for developing breast cancer. Although dry bean consumption appeared to modify cell proliferation, our findings strongly implicate apoptosis as the prominent mechanism accounting for the cancer inhibitory activity of dry bean. Whether this response is a direct consequence of the modulation of plasma concentrations of growth factors and cytokines by dry bean consumption or is secondary to the effects of small molecule anticancer compounds in small red dry bean is a question that requires further investigation.

	ACKNOWLEDGMENTS

 
We thank Leslie Brick, Vanessa Fitzgerald, Elizabeth Neil, Denise Rush, and Jennifer Sells for their excellent technical assistance.

	FOOTNOTES

 
¹ Supported in part by United States Agency for International Development grant no. REE-A-00-03-00094-00, the Bean Health Alliance, and the Colorado State University Crops for Health Research Program.

² The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

³ Author disclosures: M. D. Thompson, H. J. Thompson, M. A. Brick, J. N. McGinley, W. Jiang, Z. Zhu, and P. Wolfe, no conflicts of interest.

⁷ Abbreviations used: AC, adenocarcinoma; APAF1, anti-apoptosis protease-activating factor 1; BAX, BCL-2–associated X protein; BCL-2, B cell lymphoma 2; CRP, C-reactive protein; E2F1, E2F transcription factor 1; IGF-1, insulin growth factor-1; IL-6, interleukin-6; HRP, horseradish peroxidase; pRb, hypo-phosphorylated retinoblastoma protein; ppRb, hyper-phosphorylated retinoblastoma protein; Rb, retinoblastoma; XIAP, X-linked inhibitor of apoptosis protein.

Manuscript received 15 June 2008. Initial review completed 30 June 2008. Revision accepted 6 August 2008.

	LITERATURE CITED

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Thompson, M. D. Articles by Wolfe, P. Search for Related Content PubMed PubMed Citation Articles by Thompson, M. D. Articles by Wolfe, P.