INHIBITORY EFFECT OF PEPTIDE PREPARED FROM CORN GLUTEN MEAL ON 7,12-DIMETHYLBENZ[a]ANTHRACENE-INDUCED MAMMARY TUMOR PROGRESSION IN RATS

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Research, u Vol.17,No.7t ,pp.1121-1130,1997 r Copyright 01997EtseviaScience Inc. Printed intheUSA.Allrighw rewved 0271-5317/97 $17.00+Ml

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INHIBITORY EFFECT OF PEPTIDE PREPARED FROM CORN GLUTEN MEAL. ON 7,12 -DIMETHYLBEN.Z[a] ANTHRACENE-INDUCED MAMMARY TUMOR PROGRESSION IN RATS Magoichi Yamaguchi,Ph.D.1, Masayasu Takeuchi, Ph.D.l and Kiyoshi Ebihara, Ph.D.z I Nihon Shokuhin Kako Co., Ltd., Fuji 417, Japan z Faculty of Agriculture, Ehime University, Matsuyama 790, Japan

ABSTRACT prepared from corn gluten meal (CP) by The effect of a peptide proteolysis with alkaline protease on 7,1 2-dimethylbenz[ a]anthracene (DMBA)-induced mammary tumor progression were investigated in female Sprague-Dawley rats. At 6 weeks of age, rats were allowed free access to one of the four diets, 20% casein (CAS), 15% casein + 5%0CP (5-CP), 10% casein + 10% CP (10-CP) or 10% casein + 10% free amino acid mixture simulated CP (AA) for 10 weeks. Rats were given 5 mg DMBAin 0.5 ml corn oil by stomach tube at two times (day 7 and 21). Tumor incidence was significantly lower in the 1O-CPgroup, but not in the 5-CP and AA groups, than the CAS group. Total number and total weight of tumors were greater in the CAS group compared with 5-CP, 1O-CP or AA groups. No changes were observed in serum 17& estradiol concentrations among the three groups (CAS, 1O-CP and AA). @1997SlSeviascimc0tnc. KEYWORDS: Corn peptide, 7,12-Dimethylbenz[a] anthracene(DMBA), Mammary tumor, 17p-Estradiol, Rat INTRODUCTION Breast cancer is one of the most common cancers in Westernized countries but its incidence is significantly less in Third World and Asian populations. It is believed that some of disparity in breast cancer risk between Westernized and Asian countries reflects hormonal status, especially that of estrogen (1). Goldin et al. (2) showed that fecal estrogen excretion of Oriental women was twice as high as that of Caucasians while the plasma estrogen concentrations in the latter were higher than in Orientals. Epidemiological data from immigrant studies suggest that in most cases the susceptibility to breast cancer is the result of environmental rather than genetic It has differences between these populations and diets is a major contributing factor. Correspondence:KiyoshiEbihara, Departmentof BiologicalResources,Faculty of Agriculture,Ehime University,Matsuyama790, Japan 1121

i

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M. YAMAGUCHIet al.

been shown that high fat and high caloric intakes were associated with an increased risk for breast cancer (3). On the other hand, it has been also shown that high intakes of animal protein and red meat were associated with increased risk for breast cancer in the premenopausal women. Most of the experimental study on diet in relation to hormone-dependent cancers has been concerned with mammary tumors in mice or rats. Experimental studies support the importance of protein source and Hawrylewicz et al. (4) have reported that the incidence of mammary tumors induced by N-nitrosomethylurea was lower in rats fed a diet containing soybean protein compared with those fed a diet containing casein. A protective effect of soybean protein was recorded also by Barnes et al. (5) who noted that the number of palpable mammary tumors induced by 7, 12-dimethylbenz[ a]anthracene (DMBA) was lower in rats fed with crude or purified soybean proteins compared with those fed casein. More recently, attention has been paid for the possible protective role of plant proteins in diet-related cancers. So, we studied whether corn peptide prepared from corn gluten had an inhibitory effect on mammary tumor progression in female rats exposed to DMBA. MATERIALS AND METHODS Corn peptide (CP) was prepared from corn gluten meal by Corn peptide (CP) proteolysis with alkaline protease from alkapholic BacillusA-7 (6). Free amino acids were not found in the CP product. The molecular weight distribution of the CP was less than about 2,000, characteristic of dipeptides to decapeptides (mostly dipeptide and tripeptide). The composition of the CP was as follows (in wt%): moisture, 3.5; peptide, 92.5; and others, 4.0. The CP was somewhat similar in amino acid pattern to corn gluten meal, which was rich in glutamic acid (glutamine), alanine, proline and Ieucine, but rather poor in sulfur and basic amino acid (Table 1). Female rats (35 days of age) of the Sprague-Dawley strain Animals and Diets (purchased from Clea Japan, Inc., Osaka, Japan) were used in all experiments. They were housed in individual cages with screen-bottoms of stainless steel in a room of The animals controlled temperature (23 t l°C) and lighting (lights on: 0700-1900 h). were maintained on a commercial diet (MF@, Oriental Yeast, Tokyo) for 7 days and They were then divided into 3 groups and fed the experimental diets ad libitum. allowed free access to water. Body weight and food intake were recorded daily in the morning before replenishing the diet. The composition of experimental diets is shown in Table 2. Casein, CP and amino acid mixture simulated CP were used as protein sources. Each experimental diet contains the same level of protein. The rats received 5 mg DMBA (Nakarai Tumor induction and assessment Tesque, Inc., Kyoto, Japan) in 0.5 ml of corn oil by a stomach tube at two times (day 7 and 21 ) after the dietary transfer. In Experiment 1, rats were examined weekly for palpable mammary tumors once a week. Ten weeks after the dietary transfer, rats were killed. Following midline ventral incision, blunt dissection was used to reflect the .- —,. n--, .,-- ,.C ,.11 —,. —----...— ..-. . . .. . . ..,.. -,4 --A ,..,,.: --,4 TL :A:. .: A. .-1 ... —----the susceptibility to breast cancer is the result of environmental rather than genetic It has differences between these populations and diets is a major contributing factor. Correspondence:KiyoshiEbihara, Departmentof BiologicalResources,Faculty of Agriculture,Ehime University,Matsuyama790, Japan 1121

CORN AND MAMMARYTUMOR perpendicular W/2 X r (8).

diameter (W). Tumors

areas were calculated

1123 using the formula L/2 X

Forty five rats (42 days of age) weighing 135 g (pooled SE=2) were Experiment 1 divided into 3 group of 15 rats and allowed free access for 10 weeks to one of the three At the end of experimental period, rats diets (Diet CAS, Diet 5-CP or Diet 1O-CP,Table 2). were killed by ether exposure. Experiment 2 Forty five rats (42 days of age) weighing 130 g (pooled SE=2) were divided into 3 group of 15 rats and allowed free access for 10 weeks to one of the three diets (Diet CAS, Diet 1O-CP or Diet AA, Table 1). On the last day of feeding, food was withdrawn at 10:00 h and blood was collected in a heparinized tube from the abdominal aorta under anesthesia with Nembutal (sodium pentobarbital, Abbott Laboratories, North Chicago, IL). Amino acids and estradiol-17p were assayed on serum prepared from aortic blood. Analysis Serum (0.5 ml) was deproteinized by addition of 1 ml of S-sulfosalicylic acid (50 g/L) and centrifugated at 15,000xg for 15 min. Aliquots of the resulting supernatant were filtered through a milipore filter (0.45 mm) and amino acid concentrations were determined using an automatic amino acid analyzer (Model 835-50, Hitachi, Tokyo, Japan). Estradiol-17P was assayed by radioimmunoassay using the method of Butcher et d. (9). Statistical methods The statistical differences in tumor incidence between groups were determined by chi-square analysis with Yates correction (10). Other statistical differences ainong groups were determined by ANOVA and Duncan’s new multiple range test (11 ). Differences were considered significant at 5% level.

TABLE1 AminoAcidCompositionfor Caseinand CornPeptide(CP) Aminoacid

Isoleucine Leucine Valine Lysine Methionine Tryptophan Phenylalanine Threonine Histidine

Casein

CP

32.5 78.8 42.1 61.3 24.8 10.4 40.5 46.5 19.0

25.8 127.5 31.4 13.4 19.3 0.0 29.7 41.3 12.9

Aminoacid

Arginine Alanine Asparaticacid Cysteine Glutamicacid Glycine Proline Serine Tyrosine

Casein

28.3 26.1 51.6 5.0 165.6 15.2 74.7 48.8 46.5

CP

22.8 90.1 52.5 5.8 187.6 23.0 79.9 56.1 41.3

M. YAMAGUCHIet al.

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TABLE2 Compositionof ExperimentalDiets Constitute

DietCM

Diet5-CP

Diet1O-CP

DietAA

g/kg diet Caseinl Cornpeptidez Aminoacid mix.~ Lard Cornoil Vitamin mix.4 Mineralmix.1 Sudrose

200

150 46.5

100 93

50 50 10 40 650

50 50 10 40 653.5

50 50 10 40 657

100 92.53 50 50 10 40 657.5

1 N=13.8%. 2 N=14.8%. 3 Aminoacid mixturesimulatedcorn peptidewasaddedto the DietAAthe same levelof nitrogenas that of Diet1O-CP. 4 AmericanInstituteof Nutrition(7).

RESULTS Experiment 1 Body weight gain and food intake were unaffected by treatment (Table 3). The cumulative incidence of palpable mammary tumor in rats is shown in Figure 1. The 1O-CPdiet just slowed down the rate of tumor incidence. At the termination of the the tumor incidence was significantly lower in the 10-CP group experiment (p
DISCUSSION The role of dietary and environmental factors in cancer risk is accepted. Caloric restriction has a general inhibitory influence on tumorigenesis ( 12). High fat intake

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TABLE3 Effect of Corn Peptide(CP) on Food Intake,BodyWeightGain,Tumor Incidence,Total TumorNumber,TotalTumorWeightandTumorSizein RatsAdministeredDMBA Tumor Tumorsize Food Bodywt. incidence TotalNo. Totaltumor— intake gain Group of tumor weight <0.5 cmz 0.5 cmz< % n g/10 wks/rat Experiment 1 CAS 1044* 31a,2 153*7a 5-CP3 1029f24a 147*6a 1O-CP 1023 +25a 149 +Sa Experiment 2 CAS l137*33a 147*4 1O-CP 1081 *24a 143*7a AA 1119*19’ 141*5a

No./group g/group

No./group

15/15a 100 12/15’ 80 9/15b 60

62 46 32

35.6 18.3 8.2

49 36 29

13 7 3

14/15a 8/15b 11/15a

56 30 35

33.9 8.3 11.8

46 28 31

10 2 4

93 53 73

1 Casein. 2 Mean*SEM(n=15), values not sharing a commonsuperscript letter are significantlydifferent (p
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compared with that in CASdiet, 0.62 and 1.27%, respectively. Methionine and ar-ginine are the precursor molecules for polyamine synthesis. Decreased levels of dietary methionine and arginine may decrease polyamine synthesis and tumor cell Soy protein is low in methionine. In this study, methionine and proliferation. arginine contents in CAS and 1O-CP diets were almost the same, 4.97 and 4.41 g/kg diet for methionine and 5.66 and 5.11 g/kg diet for arginine, respectively. Schaur et al. (19) have reported that the continuous administration of physiological doses of the branched-chain amino acid (BCM) Ieucine, isoleucine and valine to Yoshida sarcomabearing rats caused a significant increase in the survival time and a significant reduction of tumor size. Crosby et al. (20) found the administration of a total parenteral nutrition (TPN) solution enriched with BCM (50% by weight) to Yoshida sarcomabearing rats increased tumor breakdown rates, but not 19Y0 BCAA TPN solution. These works have suggested that BCM-enriched TPN solution may play a role i n retarding tumor growth by increasing tumor protein breakdown rates. However, in this study, BCM per cent (by weight) in the CAS and 1O-CP diets were almost the same, 18.8 and 20.1%, respectively. DMBA-induced mammary tumors are primarily prolactin- and estrogen-dependent. It has been known that arginine increases the serum levels of prolactin, growth Salter et al. (23) found that insulin and hormone, insulin and glucagon (21,22). the growth of hormone-dependent glttcagon suppressed cancer, Walker 256

1oo- ~

CAS

80- ----0----

5-CP

604020o-

DMBA 1 [ I I I I I I I I 0 1 2 3 4 5 6 7 8 9 10

o

10

Week

❑

CAS

20

30

40

50

Estradiol-17~ (pghnl) W

1O-CP

❑

AA

Fig. 1 (left) Cumulativepalpablemammarytumor incidencein rats fed on the CAS, 5CP or 1O-CPdiet. Diet groups: CAS,20% casein; 5-CP, 15’%casein + 5% corn peptide; 1O-CP,10% casein + 10% corn peptide. Fig. 2 (right) Serumestradiol-17~concentrationsin rats fed on the CAS,1O-CPor AA diet. Diet groups: CAS,20% casein; 1O-CP,10% casein + 10% corn peptide; AA, 10% casein + 10% aminoacid mixturesimulatedcorn peptide.

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Ile Leu

Val 2;

Cys Phe Tyr Thr Trp Arg His Ala Asp Glu Ser

o

0.5

0.25

0.75

Serum free amino acid (pmol)

❑

CAS

❑

1O-CP

❑

AA

Fig. 3 Free amino acid in the serum in rats fed on CAS,1O-CPor AAdiet. Diet groups: CAS,20% casein; 1O-CP,10% casein + 10% corn peptide; AA, 10% casein + 10% amino acid mixture simulated corn peptide. * Differs significantly from CAS and 1O-CP, p-d.os. carcinosarcoma. We did not measure serum insulin, glucagon and prolactin. Takeda has reported that arginine has an inhibitory effect on growth of rat mammary tumors induced by DMBA (24). However, arginine content in the CAS and 1O-CP diets were almost the same, 5.66 and 4.95 g/kg diet, respectively. The composition of the 1O-CP and AA diets are the same, except that nitrogen source is peptide or the corresponding amino acid mixture, respectively. However, the progression of DMBA-induced mammary tumor was lower in rats fed the 1O-CPdiet, but This difference may not in rats fed the AA diet, than in rats fed the CAS diet. analogically account for possible differences in nutritional quality between peptide or the corresponding amino acid mixture. It is generally accepted that intestinal absorption of dipeptides or tripeptides during the transport process is different from that of free amino acids, and peptides are more effective in the provision of nutrition than amino acids or protein on particular occasion (25). In this study, amino acid composition may not be as important as amino acid chain length (ie dipeptide, tripeptide, etc) in the diet. Serum concentration of alanine in rats fed the AA diet was significantly higher than that in rats fed the 1O-CPdiet. BCAAprovide amino groups to produce alanine for hep~tic gluconeogenesis. In patients with actively growing neoplasms, plasma amino However, as shown acid profiles are characterized by reduced BCAA levels (26).

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M. YAMAGUCHIet al.

Figure 3, the reduction of BCAA concentrations in serum was not found in this study. Moreover there is no significant difference between serum concentrations of alanine in rsts fed the CAS and 1O-CPdiets. There were no significant differences in serum estradiol-17 p concentrations between the CAS and 1O-CP groups (Fig. 2). Thus, the present data indicate that inhibitory potency of 1O-CP on DMBA-induced mammary tumor progression is not necessary paralleled with its lowering effect of estradiol-17@ The present data showed that the difference in quality of dietary protein, namely the difference of amino acid composition, affected the progression of mammary tumor induced by a model carcinogen, DMBA. However, further studies are needed to clarify the mechanisms of inhibitory potency of dietary CP on DMBA-induced mammary tumor progression. REFERENCE 1

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