- Email: [email protected]
Inhibitory effect of soybean hypocotyls on Epstein–Barr virus early antigen induction and skin tumor promotion
Cancer Letters 121 (1997) 53–57
Inhibitory effect of soybean hypocotyls on Epstein–Barr virus early antigen induction and skin tumor promotion Yukihiro Zaizen a, Harukuni Tokuda b, Hoyoku Nishino b, Masazumi Takeshita a ,* b
a Department of Biochemistry, Oita Medical University, Hasama-machi, Oita 879-55, Japan Department of Biochemistry, Kyoto Prefectural University of Medicine, Kawaramachi-dori, Kamigyo-ku, Kyoto 602, Japan
Received 8 May 1997; accepted 23 June 1997
Abstract The in vitro anti-tumor promoting effect of hypocotyls from fresh soybeans was evaluated. The dimethyl sulfoxide extracts of hypocotyls showed a stronger inhibitory effect than that of soybeans on Epstein–Barr virus early antigen activation induced by 12-O-tetradecanoylphorbol-13-acetate. Daidzin, an isoflavone with the highest content of hypocotyls, was also inhibitory. An in vivo evaluation of anti-tumor promoting activity of hypocotyls against the skin of mice also revealed a significant inhibitory effect on tumor formation. 1997 Elsevier Science Ireland Ltd. Keywords: Soybean hypocotyl; Anti-tumor promoting effect; Epstein–Barr virus early antigen; Mouse skin tumor; Isoflavonoid
1. Introduction
2. Materials and methods
It has been suggested that soy intake may contribute to relatively low rates of breast, colon and prostate cancers. Soybeans are dietary sources of isoflavones which have been shown to have protective effects for some cancers [1]. Soybean hypocotyls are the by-product of soybean processed foods and contain a higher amount of isoflavones than soybeans [2]. In the present study, the soybean hypocotyls were tested for their effect on Epstein–Barr virus early antigen (EBV-EA) activation [3] and also analyzed for antitumor promoting activity against mice skin tumors [4].
2.1. Cell cultures
* Corresponding author. Tel.: +81 975 865670; fax: +81 975 496302; e-mail: [email protected]
Raji cell is an EBV genome-carrying human B-cell line which originated from a Burkitt’s lymphoma (BL) patient. The cells were cultured at 37°C in RPMI 1640 medium (Nipro, Osaka, Japan) supplemented with 10% fetal calf serum (FCS) (GIBCO, Australia) in a humidified atmosphere containing 5% CO2. 2.2. Test products The powdered soybean hypocotyls (raw and roast) derived from fresh soybeans and soybeans with fat removed were provided by Fuji Oil (Osaka, Japan).
0304-3835/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0335-2
54
Y. Zaizen et al. / Cancer Letters 121 (1997) 53–57
Materials (10 mg) were extracted overnight at room temperature with 1 ml dimethyl sulfoxide (DMSO) (Wako, Osaka, Japan). Isoflavonoid (daidzin, 10 mg) was dissolved in 1 ml DMSO. To make 100 mg/ml of test substance, 100 ml of the extract was transferred to 1 ml of the medium. In the same way, 10 ml of the extract was transferred. Concentration of isoflavonoids in the hypocotyls as summarized in Table 1 were analyzed by K. Takamatsu, Applied Research Institute, Fuji Oil. Isoflavonoids were extracted with methanol and were separated on reversed-phase highpressure liquid chromatography. 2.3. In vitro bioassay The activity of soybean-derived materials was assayed by inhibition of EBV-EA induction as described previously [5–7]. In brief, Raji cells were grown to a density of 1 × 106 cells/ml, harvested by centrifugation and resuspended in RPMI medium with 8% FCS containing 4 mM n-butyric acid (Sigma, St. Louis, MO, USA), 32 nM 12-O-tetradecanoylphorbol-13-acetate (TPA, Sigma) and 100 or 10 mg/ml of the test substances (DMSO extracts). The cells were incubated at 37°C for 48 h. Cell number and viability were determined after 48 h by hemocytometer counting (Trypan Blue exclusion test) [8]. The incubation was stopped by centrifugation and the cells were washed twice with 1 ml of phosphate-buffered saline (PBS), smeared on slide glass, air-dried and fixed with acetone for 10 min. The EA was stained with EA antibody (+) serum (dilution 1:120) from a patient with nasopharyngeal carcinoma (NPC) and finally checked by indirect immunofluorescence with the use of a fluorescein isothiocyanate (FITC)-conjugated goat anti-human-IgG (dilution 1:20; Medical & Biological Laboratories, Nagoya, Japan). Untreated cultures served as the controls. EBV-EA inhibiting Table 1 Isoflavonoid content (mg/g dry matter) of soybean hypocotyls Isoflavonoid
Raw hypocotyl
Roast hypocotyl
Daidzin Genistin Daizein Genistein Total
7715 1342 143 34 9234
4805 1180 1621 338 7944
activity of the test compound was estimated on the basis of the percentage of the number of positive cells compared to that of a control without the test product. In each assay, at least 500 cells were counted and the results were read blind. 2.4. The test on mice skin tumor promotion A total of 45 female ICR mice were used in this experiment. Basal diet (Oriental MF, Oriental Yeast, Tokyo) and tap water were available ad libitum throughout the experiment. The animals in groups 1–3, each consisting of 15 animals housed five per cage, were painted with dimethylbenz[a]anthracene (DMBA) on the shaved skin on the back at a quantity of 390 nmol in acetone per mouse. After 1 week, the mice were treated topically with 1.7 nmol of TPA twice a week for 20 weeks. Hypocotyls (10 mg) (raw and roast) were extracted in 1 ml DMSO and 50 ml of the extract was dissolved in 1 ml of acetone. One hour prior to TPA treatment, the animals in groups 1 and 2 were treated with 0.1 ml of 500 mg/ ml of the hypocotyls in acetone. Animals in group 3 were treated with acetone alone, serving as a promotion-positive control. The volume of inhibitors used in the present study was determined based on the previous experiments in mice [4,9]. The promoter resulted in the growth of many small benign warlike tumors called papilloma in the region that had previously been exposed to the initiator. The mice were observed weekly and the number of papillomas were counted.
3. Results The principal results of anti-tumor promoting activity of the extracts of materials are summarized in Table 2. None of the two materials examined were toxic to culture cells under these conditions as judged from cell viability. Although all extracts had an inhibitory effect on EBV-EA induction, isoflavone (daidzin), which is the major component of the extract of soybean hypocotyl, was the most active and the extract of soybean with fat removed was the least active. Although the possible involvement of other minor component cannot be excluded, these data suggest that the inhibitory effect of hypocotyl on EBV-
55
Y. Zaizen et al. / Cancer Letters 121 (1997) 53–57 Table 2 Inhibitory effect of the extracts of hypocotyls, soybean and daidzin on TPA-induced EBV-EA Test substancesd
Percentage of EBV-EA positive cellsa (% of viabilityb) Concentration of test substances (mg/ml)c 100
Hypocotyl (raw) Hypocotyl (roast) Defatted soybean Daidzin
10
TPA(−)
TPA(+)
TPA(−)
TPA(+)e
0 1 0 0
0 0 17 16
0 0 0 0
17 23 32 15
(60) (60) (60) (60)
e
(60) (60) (.70) (.70)
(.70) (.70) (.70) (.70)
(.70) (.70) (.70) (.70)
a
Values represent the means of three separate experiments. Values in parentheses are percentages of viable Raji cells. Detailed description given in Section 2. d The control without test substances showed less than 1% of EBV-EA positive cells in the absence of TPA and more than 70% viability of the cells. In the presence of TPA, EBV-EA positive cells were 34% of the total cells and the cell viability was about 60%. e TPA (+) indicates that 32 nM of TPA was added to the medium. b c
EA induction is mainly due to a result of the function of isoflavone. The hypocotyls were also studied for in vivo antitumor promoting effect using skin tumor induction in mice. As shown in Fig. 1, treatment with hypocotyls delayed the TPA-induced promotion of mouse skin papillomas. Papillomas first appeared 6 weeks after the start of promotion in the control group, while the tumors were observed 8 and 9 weeks after the first TPA treatment in raw hypocotyl and roast hypocotyl treated mice (Fig. 1A). At the end of this experiment, the mean number of papilloma was approximately 10 in the control group and five in the hypocotyl groups (Fig. 1B).
4. Discussion The results of this study indicate that both DMSO extracts from raw and roast soy hypocotyls showed inhibition of EBV-EA activation. The effect was stronger than the preparation from soybeans. Moreover, the treatment of mouse skin by the preparation of hypocotyls showed a delay of TPA-induced promotion of papillomas. As the hypocotyls include five times more isoflavones than soybeans, the inhibitory effect might come mainly from isoflavones. Numerous papers have suggested a protective effect of soy products against cancer promotion [10–15]. These include effects of isoflavones, trypsin inhibitors, phy-
tic acid and saponins. Isoflavones have a suppressive effect on proliferation of lymphocytes and many tumor cell lines in culture [16]. Quercetin and the other flavonoids are the inhibitors of protein tyrosine kinases (PTK) [17] and topoisomerases [16]. Consequently, genistein induces G2-M arrest [18] and other flavonoids (quercetin, luteolin and flavone) induce G1 arrest [20]. Furthermore, flavonoids are potent in vitro and in vivo antioxidants [19,20]. Soybean and its products are known to contain trypsin inhibitors, phytic acid and saponins other than isoflavones. Defatted flour of soybeans contains in the range of 1–2% phytic acid, whereas hypocotyl contains only 0.88% [14]. Soybeans contain 0.1– 0.5% saponins, whereas the level in the hypocotyl approaches 2% [14]. Saponin in the diet is known to reduce blood cholesterol levels [15], while in vitro saponin inhibits EBV-EA expression [21]. Although our data suggest that the anti-tumor activity is attributable to isoflavones (Table 2), the synergistic effect of isoflavones and saponin could not be neglected. Furthermore, trypsin secreted from the pancreas can cause inflammation and provoke ulcers and cancer in digestive tracts, suggesting that chronic inflammation is one of the risk factors of carcinogenesis. An inhibitory effect of prostaglandin synthesis inhibitors (aspirin and indomethacin) on carcinogenesis has been demonstrated to suggest that cancer promotion is partly due to inflammation [22]. In the present study, the activity of trypsin inhibitor was lost in the
56
Y. Zaizen et al. / Cancer Letters 121 (1997) 53–57
Fig. 1. Effect of hypocotyls on TPA-induced papillomas. (A) The number of animals having papillomas. (B) The number of papillomas occurring in each animal. (A) TPA (1.7 nmol); (S) TPA (1.7 nmol) with raw hypocotyl (50 mg); (W) TPA (1.7 nmol) with roast hypocotyl (50 mg).
roast hypocotyls due to denaturation of the inhibitor protein (data not shown). On the other hand, the inhibitory effect on EBV-EA activation and skin papilloma induction remained in the roast hypocotyls. These results indicate that the inhibition of EA induction and tumor promotion by roast hypocotyls may not be caused by trypsin inhibitors. Though isoflavones are the major substances which have been thought to reduce the risk of cancer by hypocotyls, further studies of other components that may have effects on EBV-EA expression and antitumor promotion will be necessary to understand the characteristics of inhibitory action.
Acknowledgements The authors are grateful to Dr Komei Shirabe (Department of Biochemistry, Oita Medical University) for critical reading and helpful suggestions. We would especially like to thank Dr Kiyoharu Takamatsu (Applied Research Institute, Fuji Oil) for the measurement of isoflavonoids and the assay for trypsin inhibitor and Dr Hiroshi Ueda (Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center) for his invaluable advice and support. We wish to thank Fuji Oil for the supply of experimental materials and financial support during the course of this work. Thanks are
also due to The Soy Protein Research Committee (Japan) for their support. This study was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (for Scientific Research) and from the Ministry of Health and Welfare for the Second Term Comprehensive 10-Year Strategy for Cancer Control, Japan.
References [1] M.J. Messina, V. Perskey, K.D.R. Setchell, S. Barnes, Soy intake and cancer risk: a review of the in vitro and in vivo data, Nutr. Cancer 21 (1994) 113–131. [2] L. Coward, N.C. Barnes, K.D.R. Setchell, S. Barnes, Genistein, daizein, and their b-glycoside conjugates: antitumor isoflavones in soybean foods from American and Asian diets, J. Agric. Food Chem. 41 (1993) 1961–1967. [3] G.J. Kapadia, S.C. Sharma, H. Tokuda, H. Nishino, S. Ueda, Inhibitory effect of iridoids on Epstein–Barr virus activation by a short-term in vitro assay for antitumor promoters, Cancer Lett. 102 (1996) 223–226. [4] A. Leder, A. Kuo, R.D. Cardiff, E. Sinn, P. Leder, v-Ha-ras transgene abrogates the initiation step in mouse skin tumorigenesis: effects of phorbol esters and retinoic acid, Proc. Natl. Acad. Sci. USA 87 (1990) 9178–9182. [5] Y. Ito, M. Kawanishi, T. Harayama, S. Takabayashi, Combined effect of the extracts from Croton tiglium, Euphorbia Lathyris or Euphorbia Tirucalli and n-butyrate on Epstein– Barr virus expression in human lymphoblastoid P3HR-1 and Raji cells, Cancer Lett. 12 (1981) 175–180. [6] Y. Ito, S. Yanase, J. Fujita, T. Harayama, M. Takashima, H.
Y. Zaizen et al. / Cancer Letters 121 (1997) 53–57
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
Iwanaka, A short term in vitro assay for promoter substances using human lymphoblastoid cells latently infected with Epstein–Barr virus, Cancer Lett. 13 (1981) 29–37. I. Honda, H. Tokuda, M. Kozuka, K. Yoneyama, H. Nishino, A. Iwashima, M. Shibagaki, M. Noma, N. Takahashi, S. Yoshida, Inhibitory effects of 3-nitro-2,4,6-trihydroxybenzamides on Epstein–Barr virus early antigen induction, Cancer Lett. 59 (1991) 83–88. K.K. Sanford, W.R. Earle, V.J. Evans, H.K. Waltz, J.E. Shannon, The measurement of proliferation in tissue cultures by enumeration of cell nuclei, J. Natl. Cancer Inst. 11 (1951) 773–795. G.J. Kapadia, H. Tokuda, T. Konoshima, H. Nishino, Chemoprevention of lung and skin cancer by Beta vulgaris (beet) root extract, Cancer Lett. 100 (1996) 211–214. L.W. Wattenberg, J.L. Leong, Inhibition of the carcinogenic action of benzo[a]pyrene by flavones, Cancer Res. 30 (1970) 1922–1925. L. Diamond, R. MacFall, J. Miller, V. Gelboin, The effects of two isomeric benzoflavones on aryl hydrocarbon hydroxylase and the toxicity and carcinogenicity of polycyclic hydrocarbons, Cancer Res. 32 (1972) 731–736. H. Nishino, A. Iwashima, H. Fujiki, T. Sugimura, Inhibition by quercetin of the promoting effect of teleocidin on skin papilloma formation in mice initiated with 7,12,-dimethylbenz[a]anthracene, Gann 75 (1984) 113–116. T. Fotsis, M. Pepper, H. Adlercreutz, T. Hase, R. Montesano, L. Schweigerer, Genistein, a dietary ingested isoflavonoid, inhibits cell proliferation and in vitro angiogenesis, J. Nutr. 125 (1995) 790–797. R.L. Anderson, W.J. Wolf, Compositional changes in trypsin
[15] [16]
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[18]
[19]
[20]
[21]
[22]
57
inhibitors, phytic acid, saponins and isoflavones related to soybean processing, J. Nutr. 125 (1995) 581S–588S. M. Messina, S. Barnes, The role of soy products in reducing risk of cancer, J. Natl. Cancer Inst. 83 (1991) 541–546. K. Kondo, K. Tsuneizumi, T. Watanabe, M. Oishi, Induction of in vitro differentiation of mouse embryonal carcinoma (F9) cells by inhibitors of topoisomerases, Cancer Res. 51 (1991) 5398–5404. T. Akiyama, J. Ishida, S. Nakagawa, H. Ogawara, S. Watanabe, N. Itoh, M. Shibuya, Y. Fukami, Genistein, a specific inhibitor of tyrosine-specific protein kinases, J. Biol. Chem. 262 (1987) 5592–5595. Y. Matsukawa, N. Marui, T. Sakai, Y. Satomi, M. Yoshida, K. Matsumoto, H. Nishino, A. Aoike, Genistein arrests cell cycle progression at G2-M, Cancer Res. 53 (1993) 1328– 1331. C.V. de Whalley, S.M. Rankin, J.R.S. Hoult, W. Jessup, D.S. Leake, Flavonoids inhibit the oxidative modification of low density lipoproteins by macrophages, Biochem. Pharmacol. 39 (1990) 1743–1750. K. Ioku, T. Tsushida, Y. Takei, N. Nakatani, J. Terao, Antioxidative activity of quercetin and quercetin monoglucosides in solution and phospholipid bilayers, Biochem. Biophys. Acta 1234 (1995) 99–104. H. Tokuda, T. Konoshima, M. Kozuka, T. Kimura, Inhibitory effects of 12-O-tetradecanoylphorbol-13-acetate and teleocidin B induced Epstein–Barr virus by saponin and its related compounds, Cancer Lett. 40 (1988) 309–317. L.J. Marnett, Aspirin and the potential role of prostaglandins in colon cancer, Cancer Res. 52 (1992) 5575–5589.
Inhibitory effect of soybean hypocotyls on Epstein–Barr virus early antigen induction and skin tumor promotion Yukihiro Zaizen a, Harukuni Tokuda b, Hoyoku Nishino b, Masazumi Takeshita a ,* b
a Department of Biochemistry, Oita Medical University, Hasama-machi, Oita 879-55, Japan Department of Biochemistry, Kyoto Prefectural University of Medicine, Kawaramachi-dori, Kamigyo-ku, Kyoto 602, Japan
Received 8 May 1997; accepted 23 June 1997
Abstract The in vitro anti-tumor promoting effect of hypocotyls from fresh soybeans was evaluated. The dimethyl sulfoxide extracts of hypocotyls showed a stronger inhibitory effect than that of soybeans on Epstein–Barr virus early antigen activation induced by 12-O-tetradecanoylphorbol-13-acetate. Daidzin, an isoflavone with the highest content of hypocotyls, was also inhibitory. An in vivo evaluation of anti-tumor promoting activity of hypocotyls against the skin of mice also revealed a significant inhibitory effect on tumor formation. 1997 Elsevier Science Ireland Ltd. Keywords: Soybean hypocotyl; Anti-tumor promoting effect; Epstein–Barr virus early antigen; Mouse skin tumor; Isoflavonoid
1. Introduction
2. Materials and methods
It has been suggested that soy intake may contribute to relatively low rates of breast, colon and prostate cancers. Soybeans are dietary sources of isoflavones which have been shown to have protective effects for some cancers [1]. Soybean hypocotyls are the by-product of soybean processed foods and contain a higher amount of isoflavones than soybeans [2]. In the present study, the soybean hypocotyls were tested for their effect on Epstein–Barr virus early antigen (EBV-EA) activation [3] and also analyzed for antitumor promoting activity against mice skin tumors [4].
2.1. Cell cultures
* Corresponding author. Tel.: +81 975 865670; fax: +81 975 496302; e-mail: [email protected]
Raji cell is an EBV genome-carrying human B-cell line which originated from a Burkitt’s lymphoma (BL) patient. The cells were cultured at 37°C in RPMI 1640 medium (Nipro, Osaka, Japan) supplemented with 10% fetal calf serum (FCS) (GIBCO, Australia) in a humidified atmosphere containing 5% CO2. 2.2. Test products The powdered soybean hypocotyls (raw and roast) derived from fresh soybeans and soybeans with fat removed were provided by Fuji Oil (Osaka, Japan).
0304-3835/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0335-2
54
Y. Zaizen et al. / Cancer Letters 121 (1997) 53–57
Materials (10 mg) were extracted overnight at room temperature with 1 ml dimethyl sulfoxide (DMSO) (Wako, Osaka, Japan). Isoflavonoid (daidzin, 10 mg) was dissolved in 1 ml DMSO. To make 100 mg/ml of test substance, 100 ml of the extract was transferred to 1 ml of the medium. In the same way, 10 ml of the extract was transferred. Concentration of isoflavonoids in the hypocotyls as summarized in Table 1 were analyzed by K. Takamatsu, Applied Research Institute, Fuji Oil. Isoflavonoids were extracted with methanol and were separated on reversed-phase highpressure liquid chromatography. 2.3. In vitro bioassay The activity of soybean-derived materials was assayed by inhibition of EBV-EA induction as described previously [5–7]. In brief, Raji cells were grown to a density of 1 × 106 cells/ml, harvested by centrifugation and resuspended in RPMI medium with 8% FCS containing 4 mM n-butyric acid (Sigma, St. Louis, MO, USA), 32 nM 12-O-tetradecanoylphorbol-13-acetate (TPA, Sigma) and 100 or 10 mg/ml of the test substances (DMSO extracts). The cells were incubated at 37°C for 48 h. Cell number and viability were determined after 48 h by hemocytometer counting (Trypan Blue exclusion test) [8]. The incubation was stopped by centrifugation and the cells were washed twice with 1 ml of phosphate-buffered saline (PBS), smeared on slide glass, air-dried and fixed with acetone for 10 min. The EA was stained with EA antibody (+) serum (dilution 1:120) from a patient with nasopharyngeal carcinoma (NPC) and finally checked by indirect immunofluorescence with the use of a fluorescein isothiocyanate (FITC)-conjugated goat anti-human-IgG (dilution 1:20; Medical & Biological Laboratories, Nagoya, Japan). Untreated cultures served as the controls. EBV-EA inhibiting Table 1 Isoflavonoid content (mg/g dry matter) of soybean hypocotyls Isoflavonoid
Raw hypocotyl
Roast hypocotyl
Daidzin Genistin Daizein Genistein Total
7715 1342 143 34 9234
4805 1180 1621 338 7944
activity of the test compound was estimated on the basis of the percentage of the number of positive cells compared to that of a control without the test product. In each assay, at least 500 cells were counted and the results were read blind. 2.4. The test on mice skin tumor promotion A total of 45 female ICR mice were used in this experiment. Basal diet (Oriental MF, Oriental Yeast, Tokyo) and tap water were available ad libitum throughout the experiment. The animals in groups 1–3, each consisting of 15 animals housed five per cage, were painted with dimethylbenz[a]anthracene (DMBA) on the shaved skin on the back at a quantity of 390 nmol in acetone per mouse. After 1 week, the mice were treated topically with 1.7 nmol of TPA twice a week for 20 weeks. Hypocotyls (10 mg) (raw and roast) were extracted in 1 ml DMSO and 50 ml of the extract was dissolved in 1 ml of acetone. One hour prior to TPA treatment, the animals in groups 1 and 2 were treated with 0.1 ml of 500 mg/ ml of the hypocotyls in acetone. Animals in group 3 were treated with acetone alone, serving as a promotion-positive control. The volume of inhibitors used in the present study was determined based on the previous experiments in mice [4,9]. The promoter resulted in the growth of many small benign warlike tumors called papilloma in the region that had previously been exposed to the initiator. The mice were observed weekly and the number of papillomas were counted.
3. Results The principal results of anti-tumor promoting activity of the extracts of materials are summarized in Table 2. None of the two materials examined were toxic to culture cells under these conditions as judged from cell viability. Although all extracts had an inhibitory effect on EBV-EA induction, isoflavone (daidzin), which is the major component of the extract of soybean hypocotyl, was the most active and the extract of soybean with fat removed was the least active. Although the possible involvement of other minor component cannot be excluded, these data suggest that the inhibitory effect of hypocotyl on EBV-
55
Y. Zaizen et al. / Cancer Letters 121 (1997) 53–57 Table 2 Inhibitory effect of the extracts of hypocotyls, soybean and daidzin on TPA-induced EBV-EA Test substancesd
Percentage of EBV-EA positive cellsa (% of viabilityb) Concentration of test substances (mg/ml)c 100
Hypocotyl (raw) Hypocotyl (roast) Defatted soybean Daidzin
10
TPA(−)
TPA(+)
TPA(−)
TPA(+)e
0 1 0 0
0 0 17 16
0 0 0 0
17 23 32 15
(60) (60) (60) (60)
e
(60) (60) (.70) (.70)
(.70) (.70) (.70) (.70)
(.70) (.70) (.70) (.70)
a
Values represent the means of three separate experiments. Values in parentheses are percentages of viable Raji cells. Detailed description given in Section 2. d The control without test substances showed less than 1% of EBV-EA positive cells in the absence of TPA and more than 70% viability of the cells. In the presence of TPA, EBV-EA positive cells were 34% of the total cells and the cell viability was about 60%. e TPA (+) indicates that 32 nM of TPA was added to the medium. b c
EA induction is mainly due to a result of the function of isoflavone. The hypocotyls were also studied for in vivo antitumor promoting effect using skin tumor induction in mice. As shown in Fig. 1, treatment with hypocotyls delayed the TPA-induced promotion of mouse skin papillomas. Papillomas first appeared 6 weeks after the start of promotion in the control group, while the tumors were observed 8 and 9 weeks after the first TPA treatment in raw hypocotyl and roast hypocotyl treated mice (Fig. 1A). At the end of this experiment, the mean number of papilloma was approximately 10 in the control group and five in the hypocotyl groups (Fig. 1B).
4. Discussion The results of this study indicate that both DMSO extracts from raw and roast soy hypocotyls showed inhibition of EBV-EA activation. The effect was stronger than the preparation from soybeans. Moreover, the treatment of mouse skin by the preparation of hypocotyls showed a delay of TPA-induced promotion of papillomas. As the hypocotyls include five times more isoflavones than soybeans, the inhibitory effect might come mainly from isoflavones. Numerous papers have suggested a protective effect of soy products against cancer promotion [10–15]. These include effects of isoflavones, trypsin inhibitors, phy-
tic acid and saponins. Isoflavones have a suppressive effect on proliferation of lymphocytes and many tumor cell lines in culture [16]. Quercetin and the other flavonoids are the inhibitors of protein tyrosine kinases (PTK) [17] and topoisomerases [16]. Consequently, genistein induces G2-M arrest [18] and other flavonoids (quercetin, luteolin and flavone) induce G1 arrest [20]. Furthermore, flavonoids are potent in vitro and in vivo antioxidants [19,20]. Soybean and its products are known to contain trypsin inhibitors, phytic acid and saponins other than isoflavones. Defatted flour of soybeans contains in the range of 1–2% phytic acid, whereas hypocotyl contains only 0.88% [14]. Soybeans contain 0.1– 0.5% saponins, whereas the level in the hypocotyl approaches 2% [14]. Saponin in the diet is known to reduce blood cholesterol levels [15], while in vitro saponin inhibits EBV-EA expression [21]. Although our data suggest that the anti-tumor activity is attributable to isoflavones (Table 2), the synergistic effect of isoflavones and saponin could not be neglected. Furthermore, trypsin secreted from the pancreas can cause inflammation and provoke ulcers and cancer in digestive tracts, suggesting that chronic inflammation is one of the risk factors of carcinogenesis. An inhibitory effect of prostaglandin synthesis inhibitors (aspirin and indomethacin) on carcinogenesis has been demonstrated to suggest that cancer promotion is partly due to inflammation [22]. In the present study, the activity of trypsin inhibitor was lost in the
56
Y. Zaizen et al. / Cancer Letters 121 (1997) 53–57
Fig. 1. Effect of hypocotyls on TPA-induced papillomas. (A) The number of animals having papillomas. (B) The number of papillomas occurring in each animal. (A) TPA (1.7 nmol); (S) TPA (1.7 nmol) with raw hypocotyl (50 mg); (W) TPA (1.7 nmol) with roast hypocotyl (50 mg).
roast hypocotyls due to denaturation of the inhibitor protein (data not shown). On the other hand, the inhibitory effect on EBV-EA activation and skin papilloma induction remained in the roast hypocotyls. These results indicate that the inhibition of EA induction and tumor promotion by roast hypocotyls may not be caused by trypsin inhibitors. Though isoflavones are the major substances which have been thought to reduce the risk of cancer by hypocotyls, further studies of other components that may have effects on EBV-EA expression and antitumor promotion will be necessary to understand the characteristics of inhibitory action.
Acknowledgements The authors are grateful to Dr Komei Shirabe (Department of Biochemistry, Oita Medical University) for critical reading and helpful suggestions. We would especially like to thank Dr Kiyoharu Takamatsu (Applied Research Institute, Fuji Oil) for the measurement of isoflavonoids and the assay for trypsin inhibitor and Dr Hiroshi Ueda (Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center) for his invaluable advice and support. We wish to thank Fuji Oil for the supply of experimental materials and financial support during the course of this work. Thanks are
also due to The Soy Protein Research Committee (Japan) for their support. This study was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (for Scientific Research) and from the Ministry of Health and Welfare for the Second Term Comprehensive 10-Year Strategy for Cancer Control, Japan.
References [1] M.J. Messina, V. Perskey, K.D.R. Setchell, S. Barnes, Soy intake and cancer risk: a review of the in vitro and in vivo data, Nutr. Cancer 21 (1994) 113–131. [2] L. Coward, N.C. Barnes, K.D.R. Setchell, S. Barnes, Genistein, daizein, and their b-glycoside conjugates: antitumor isoflavones in soybean foods from American and Asian diets, J. Agric. Food Chem. 41 (1993) 1961–1967. [3] G.J. Kapadia, S.C. Sharma, H. Tokuda, H. Nishino, S. Ueda, Inhibitory effect of iridoids on Epstein–Barr virus activation by a short-term in vitro assay for antitumor promoters, Cancer Lett. 102 (1996) 223–226. [4] A. Leder, A. Kuo, R.D. Cardiff, E. Sinn, P. Leder, v-Ha-ras transgene abrogates the initiation step in mouse skin tumorigenesis: effects of phorbol esters and retinoic acid, Proc. Natl. Acad. Sci. USA 87 (1990) 9178–9182. [5] Y. Ito, M. Kawanishi, T. Harayama, S. Takabayashi, Combined effect of the extracts from Croton tiglium, Euphorbia Lathyris or Euphorbia Tirucalli and n-butyrate on Epstein– Barr virus expression in human lymphoblastoid P3HR-1 and Raji cells, Cancer Lett. 12 (1981) 175–180. [6] Y. Ito, S. Yanase, J. Fujita, T. Harayama, M. Takashima, H.
Y. Zaizen et al. / Cancer Letters 121 (1997) 53–57
[7]
[8]
[9]
[10]
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