Clinical & Experimental Metastasis 19: 535–540, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

535

Dietary supplementation with isolated soy protein reduces metastasis ofmammary carcinoma cells in mice

Lin Yan, Donghua Li & John A. YeeDepartment of Biomedical Sciences, Creighton University School of Medicine, Omaha, Nebraska, USA

Received 3 April 2002; accepted in revised form 13 May 2002

Key words: diet, mice, mammary carcinoma, spontaneous metastasis, soy protein

Abstract

The present study investigated the effect of dietary supplementation with isolated soy protein (ISP) on pulmonary metastasisof carcinoma cells from primary mammary tumors induced by orthotopic injection of 4526 murine mammary carcinomacells in female BALB/c mice. Three diets were compared: a basal AIN-93G diet and the basal diet supplemented with10% or 20% ISP. After three weeks on the experimental diets, each mouse was injected 4 × 105 carcinoma cells into theright inguinal mammary fat pad. The primary tumors were excised when they reached a size of 1.0 cm in diameter. Aftersurgery, mice were maintained on their respective diets for another three weeks. At necropsy, the incidence of metastasis, thenumber and size of macroscopic tumors, and the number of microscopic tumors in the lungs were determined. The incidenceof mice with macroscopically visible tumors was 93%, 76%, and 67%, and the median number of macroscopic tumor was5, 2, and 1 for the control, 10%, and 20% ISP groups (P ≤ 0.05, 20% ISP vs. control). The median cross-sectional areaof the macroscopic tumors was 0.93 mm2, 0.80 mm2, and 0.31 mm2, and the median volume was 0.73 mm3, 0.56 mm3,and 0.14 mm3 for the control, 10%, and 20% ISP groups (P ≤ 0.01, 20% ISP vs. control). Histological examinationrevealed fewer microscopically detectable tumors in the ISP groups compared with the control. These results demonstratedthat dietary supplementation with ISP reduced pulmonary metastasis of carcinoma cells from primary mammary tumors inBALB/c mice.

Abbreviation: ISP – isolated soy protein

Introduction

Research on soy and breast cancer prevention has drawn at-tention in recent years in the scientific community as well asin the general public in the United States. Epidemiologicalstudies demonstrated that consumption of soy is associatedwith a lower incidence of breast cancer in Chinese women[1] and in Asian-American women in the United States [2].Soy is a major source of dietary protein in Oriental countries.That consumption of soyfoods reduces the risk of breast can-cer in humans is supported by laboratory studies that dietarysupplementation with soy preparations (e.g., isolated soyprotein (ISP)) inhibits experimentally induced mammary tu-morigenesis in animals [3, 4]. Studies also reveal that certainsoy constituents may contribute to the cancer-attenuating ef-fect of dietary soy. For example, isoflavones, a group ofdiphenolic compounds found in soy, inhibit mammary tumordevelopment in animals [5, 6].

Breast cancer is the second leading cause of deathfrom cancer in women in the United States. Each year

Correspondence to: Dr Lin Yan, Department of Nutritional Sciences, DuPont Protein Technologies, P.O. Box. 88940, St Louis, MO 63188, USA(current address). Tel: +1-314-982-1863; Fax: +1-314-982-2107; E-mail:lyan@protein.com.

there are approximately 180,000 new cases reported [7]. Atthe present time, approximately 1.7 million women in theUnited States have a history of breast cancer, and this num-ber is expected to rise to over two million in the next fiveyears [8]. Breast cancer patients and women with a historyof breast cancer are at risk of metastatic disease, includingrecurrence. Metastasis, the spread of malignant cells from aprimary tumor to a distant organ or to a different site withinthe same organ, results in the development of secondary tu-mors. Malignant spread is a major cause of death in cancerpatients. Clearly, reducing metastasis is a way to improvethe prognosis of cancer patients. Until now, the attentionof most research on diet and breast cancer has been to theprevention of primary tumor development. However, limiteddata have shown that dietary intervention may be a useful nu-tritional adjuvant in reducing the risk of metastasis in breastcancer patients. For example, it has been reported that whencombined with surgical and therapeutic treatments, dietarysupplementation with nutritional antioxidants including vi-tamin C, vitamin E, β-carotene, and selenium, reducesfurther metastasis in breast cancer patients with regionalmalignant spread to axillary lymph nodes [9]. Furthermore,laboratory studies showed that dietary supplementation withomega-3 fatty acids [10], vitamin D analogs [11], selenium

536 L. Yan et al.

[12], or soy [13] reduces experimentally induced metastasisin animals.

The objective of the present study was to determinewhether soy consumption reduces malignant spread of mam-mary carcinoma cells in mice. To accomplish this, a sponta-neous metastasis model was employed, in which the effect ofdietary supplementation with ISP on pulmonary metastasisof carcinoma cells from primary mammary tumors inducedby orthotopic injection of 4526 mammary carcinoma cellswas investigated in female BALB/c mice,

Materials and methods

Animals and diets

The protocol of the present study was reviewed and ap-proved by the Creighton University Animal Care and UseCommittee and complied with the Guide for the Care andUse of Laboratory Animals [14]. Three-week-old femaleBALB/c mice (Charles River, Wilmington, Massachusetts)were housed five per box, in wire-topped plastic boxes, in apathogen-free room and kept on a 12:12-h light–dark cycle.The temperature in the room was maintained at 25 ± 1 ◦C.During the course of the experiment mice had free accessto the diet and deionized water and were weighed weekly.Three diets were compared: a basal diet and the basal dietsupplemented with 10% or 20% ISP in place of casein. Di-etary formulations were based on the AIN-93G standard diet[15], except that soybean oil was replaced with corn oil.The ISP (Supro 670, lot #C6L-XRM-002, Protein Technolo-gies International, St. Louis, Missouri) contained 0.55 mgdaidzein, 1.17 mg genistein, and 0.10 mg glycitein on a pergram product basis. Diet components were purchased fromICN (Costa Mesa, California). All diets were prepared in ourlaboratory, and each lot was stored at 4 ◦C for no longer than3 weeks.

Mammary carcinoma cells

The 4526 murine mammary carcinoma cell line [16], ahighly metastatic cell line that spreads to the lungs, was usedin this study (provided by Dr Fred Miller, Michigan Can-cer Foundation, Detroit, Michigan). Cells were cultured inDulbecco’s minimum essential medium supplemented with5% fetal bovine serum, 5% newborn calf serum, 1 mMnonessential amino acids, and 2 mM L-glutamine. On theday of injection the cells were collected from the mono-layer culture by a brief trypsinization (0.05% trypsin and0.53 mmol/L EDTA). The viability of the cells was deter-mined with trypan blue, and a single cell suspension wasprepared in serum-free medium.

Experimental design

Ninety-eight mice were fed the basal diet for 2 days beforebeing randomly assigned to one of the three groups (36 in thecontrol group, and 31 in each of the ISP groups) that werefed either the basal diet or one of the ISP-supplemented diets

for 3 weeks. Each mouse was then administered an injectionof 4 × 105 viable carcinoma cells into the right inguinalmammary fat pad in a volume of 20 µl. To avoid possiblechanges in cell viability, cells were injected into mice within30 min after their collection from the culture. The order thatcells were injected into mice was randomized among thethree groups of animals. The occurrence and growth of pri-mary tumors were monitored on a daily basis. To avoid rapidgrowth of the primary tumor that might be a life threat to thehost, the tumors were surgically excised when they reached1.0 cm in diameter (approximately 2 weeks after the injec-tion). Mice with tumors that could not be surgically removedwere excluded from the experiment. Following removal ofthe tumor, the mice were maintained on their respective dietsfor an additional 3 weeks.

One week before tumor cell injection, six mice from eachgroup were selected at random and transferred to metaboliccages. The food intake for each mouse was recorded foreight days. The daily urine output from each animal wascollected, and samples for each group (n = 6) were pooledand analyzed for isoflavones [17].

At the end of the experiment, mice were anesthetized us-ing ketamine (50 mg kg body weight) and xylazine (5 mg kgbody weight) and then killed by cervical dislocation. Ma-jor organs were grossly examined for visible tumors. Thelungs were excised, and fixed in 10% phosphate-bufferedformalin. The number of macroscopically visible tumors ineach lung was counted using a dissecting microscope. Thecross-sectional area of tumors in randomly selected fieldswas measured using a Quantimet 500 image analysis system(Leica Cambridge, Cambridge, UK). Tumor volume wascalculated using the mean of the longest and shortest diam-eters and the assumption that tumors were spherical [18].The lungs from 10 randomly selected mice in each groupwere processed further for detection of microscopic tumors.The superior lobe from each lung was cut into small blocks,embedded in epoxy, sectioned at 2 µm in a sagittal plane,mounted on glass microscope slide, stained with toludineblue, coverslipped, and examined by light microscopy at amagnification of 40×.

Statistical analysis

The incidence of metastasis among groups was comparedusing Fisher’s exact test [19]. Bartlett’s test [20] for homo-geneity of variances showed that standard deviations for themean values of the number, cross-sectional area, and vol-ume of macroscopic tumors differed significantly among thegroups (P < 0.05). Thus, these results were analyzed usingthe Kruskal–Wallis nonparametric test and Dunn’s multiplecomparison test [21]. The data were analyzed using the sta-tistic program Instat 2.01 for Macintosh. Differences wereconsidered significant at P ≤ 0.05.

Results

Dietary supplemented with 10% or 20% ISP had no effecton body weight gain in mice compared with those fed the

Isolated soy protein and metastasis 537

Table 1. Effect of dietary supplementation with isolated soy protein on pulmonary metastasis of 4526 mammarycarcinoma cells in mice.

Group Incidence of metastasis Number of metastatic tumors developed in the lungs

Macrometastasis Micrometastasis Macrometastasis Micrometastasis

(%)a (%)b Median Mean ± SEMc tumor numbersd

Control 93 (25/27) 30 (3/10) 5 10 ± 2 8

10% ISP 76 (16/21) 20 (2/10) 2 5 ± 1 2

20% ISP 67e (14/21) 30 (3/10) 1e 4 ± 2 3

aNumber of mice bearing macroscopically visible tumors in the lungs over total mice per group.bNumber of mice with microscopic tumors detected in the superior lobe of the lungs in 10 randomly selected micefrom each group.cBecause Bartlett’s test [20] for homogeneity of variances showed heterogeneous variances among sample popula-tions, the mean values were not compared by ANOVA.dNumber of the microscopic tumors detected in the superior lobes of the lungs in 10 randomly selected mice fromeach group.eSignificantly different from the control, P ≤ 0.05. Data were analyzed using Fisher’s exact test (incidence ofmetastasis) [19] and the Kruskal–Wallis nonparametric test and Dunn’s multiple comparison test (the median valuesof macrometastasis) [21].

basal diet during the experiment (data not shown). The meanbody weight for mice at the beginning and at the end of theexperiment was 11 ± 1 g and 19 ± 1 g, respectively. Themean food intake for all mice was 2.3 ± 0.4 g/day (n =18), and there was no difference among groups (data notshown). The amount of total isoflavones (including daidzein,genistein, glycitein, equol, and o-desmethylangolensin) ex-creted in the urine was 0, 0.59 µmol/mouse/day, and1.37 µmol/mouse/day for mice fed the basal, 10%, and 20%ISP diets, respectively. Results of the necropsy showed thatthere were 17 grossly visible metastatic tumors found in ma-jor organs (kidneys, diaphragm, skin, heart, abdominal andpelvic cavities, and muscles) in the control group, whereas 2and 3 tumors were found in the 10% and 20% ISP groups.

The incidence of mice with macroscopically detectablelung tumors and the number of tumors per mouse were de-termined to assess the effect of dietary supplementation withISP on spontaneous metastasis of mammary carcinoma cellsin mice. At the end of the study, macroscopically visiblelung tumors were observed in 93% of the mice fed the basaldiet (Table 1). Feeding mice a diet containing 10% or 20%ISP resulted in a 17% or a 26% reduction in the incidenceof metastasis compared with the controls. The differencebetween the 20% ISP group and the control group was statis-tically significant (P ≤ 0.05). The median number of lungtumor in the control group was 5. Feeding mice the 10% andthe 20% ISP diets reduced the median number of tumorsto 2 and 1, respectively (Table 1). The difference betweenthe 20% ISP group and the control group was at P ≤ 0.05.The addition of ISP to the diet also reduced microscopicallydetectable tumors in mice (Table 1).

The cross-sectional area and volume of macroscopic tu-mors were measured to determine the effect of ISP dietson the growth of metastatic tumors developed in the lungs.The median cross-sectional area and volume of tumors inmice fed the basal diet were 0.93 mm2 and 0.73 mm3, re-spectively. Both cross-sectional area and volume of lungtumors in mice fed the ISP diets were decreased comparedwith the controls (Table 2). The difference between the 20%

ISP group and the control group was statistically significant(P ≤ 0.01).

Discussion

The present study was undertaken to determine whether di-etary supplementation with ISP affected spontaneous metas-tasis of mammary carcinoma cells to the lungs in mice.Injection of 4526 mammary carcinoma cells into the in-guinal mammary fat pad of young adult BALB/c femalemice resulted in the development of primary tumors at theinjection site in all the mice and macroscopically visibletumors in the lungs in 93% of the mice fed the basal diet.This demonstrated the success of the model. Results ofthe present study showed that dietary supplementation withISP reduced the incidence of mice with macroscopicallydetectable tumors as well as the number of metastatic tu-mors in the lungs. Further assessing tumor number at themicroscopic level did not eliminate these differences. Wealso found that the cross-sectional area and volume of lungtumors in mice fed the ISP diets were less than those inmice fed the basal diet, suggesting that tumors were smallerin mice that consumed an ISP diet. Furthermore, resultsof necropsy showed that there were fewer grossly visibletumors in the ISP groups than in the control group. Theseresults demonstrated that dietary supplementation with ISPprotected mice against spontaneous metastasis of mammarycarcinoma cells and reduced the growth of tumors arisenfrom malignant cells that successfully spread to the lungs.

We previously reported that dietary supplementationwith ISP reduces pulmonary metastasis of melanoma cellsfollowing the intravenous injection of melanoma cells intomice [22]. The principle advantage of this intravenous in-jection model is that it standardizes the onset of invasion bydirectly injecting neoplastic cells into the blood circulationand measures the ability of malignant cells to extravasateand form tumors in the lung. However, this model does notreflect all the events of the metastatic cascade (e.g., intrava-sation of malignant cells from a primary tumor could not be

Isolated soy protein and metastasis 539

rosine kinases [29]. Protein tyrosine kinases are involved insignal transduction events, including integrin-mediated celladhesion [30]. Genistein decreases EGF-like growth factor-stimulated integrin expression in human breast cancer cells[31] and inhibits integrin-mediated cell adhesion in lyphomacells [32]. It has been reported that genistein inhibits thesecretion of proteolytic enzymes, e.g., urokinase-type plas-minogen activator and metalloproteinase, in mammary tu-mor cells [33, 34]. Studies also revealed that genisteinhas antiangiogenic activity [35], which may play an im-portant role in inhibiting the development and growth of aneoplasm. While these studies were done using concentra-tions of genistein that may not be achievable from a regularsoy diet, they suggest possible mechanisms through whichisoflavones affect metastasis. In addition to isoflavones,other soy constituents may also contribute to the cancer at-tenuating actions. For example, a unique 43 amino acid soypeptide has been shown to inhibit tumorigenesis in a skintumor model [36]. Soy protease inhibitors [37] and saponins[38] have also been shown to have anticancer potentials. Theeffectiveness of these soy constituents at dietary levels ininhibiting neoplastic development and metastasis remains tobe investigated.

Finding ways to prevent malignant spread from a pri-mary mammary tumor would reduce the mortality of breastcancer patients. Our finding that dietary supplementationwith ISP reduces spontaneous metastasis of mammary car-cinoma cells in mice supports a growing literature showingthe benefits of soy in reducing the development of metastaticdisease. For example, it has been reported that dietary soysupplementation inhibits metastasis of breast cancer cells[39] and rhabdomyosarcoma and Lewis lung carcinoma cellsin animals [13]. A more recent study showed that dietarysupplementation with ISP is synergistic with tamoxifen ininhibiting chemically induced mammary tumorigenesis inrats [40]. Overall, these findings along with the results of thepresent study suggest that soy is a useful nutritional adjuvantin reducing the risk of metastatic disease. Future clinicalinvestigations on soy and secondary cancer prevention arewarranted.

Acknowledgements

This work was supported, in part, by grants from the Amer-ican Institute for Cancer Research (98A095) and NebraskaDepartment of Health and Human Services (99-43). The au-thors thank Ying Wu, Carole A. Miller, and Tricia Higginsfor technical assistance and Dr Patricia A. Murphy at IowaState University for isoflavone analysis.

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