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Japanese Journal of Clinical Oncology Pages 360-363

Effects of Soybean Isoflavones on Cell Growth and Apoptosis of the Human Prostatic Cancer Cell Line LNCaP
Introduction
Materials And Methods
   Chemicals
   Cell Culture
   Measurement of Proliferation Rates
   Measurement of DNA Synthesis
   Nuclear Morphology
   DNA Fragmentation Assay
   Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblot Analysis
Results
Discussion
Acknowledgments
References
Effects of Soybean Isoflavones on Cell Growth and Apoptosis of the Human Prostatic Cancer Cell Line LNCaP

Effects of Soybean Isoflavones on Cell Growth and Apoptosis of the Human Prostatic Cancer Cell Line LNCaP

Mizuki Onozawa1,2, Kazunori Fukuda1, Mikinobu Ohtani2, Hideyuki Akaza2, Takashi Sugimura1, Keiji Wakabayashi1

1Cancer Prevention Division, National Cancer Center Research Institute, Tokyo and 2Department of Urology, Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan

Background: Epidemiological studies have suggested that soybean isoflavones are associated with a lower risk of prostate cancer. However, the mechanisms of prostate cancer prevention by soybean isoflavones have yet to be fully clarified.
Methods: Two soybean isoflavones (genistein and daidzein) and their glucosides (genistin and daidzin) were tested for their effects on cell growth and apoptosis of the LNCaP human prostatic cancer cell line.
Results: Among these isoflavones, genistein was found to inhibit the growth of LNCaP most effectively, with an IC50 value of 40 µM. The inhibition of cell growth by genistein was accompanied by the suppression of DNA synthesis and the induction of apoptosis. Expression of prostate-specific antigen (PSA) in LNCaP was also significantly reduced by the treatment with genistein.
Conclusions: The results suggest that genistein might primarily influence human prostate cancer development by reducing tumor growth.

Key words: genistein - isoflavone - soybean - prostate cancer

INTRODUCTION

The incidence of prostate cancer in Japan is much lower than in Western countries (1,2). A higher consumption of soybeans and related products is speculated to be one contributory dietary factor (3-5). An inverse association between intake of soybean products and risk of cancer has also been suggested for cancers of other organs such as the breast and the colon (5,6).

The average daily consumption of soybean and its products per person in Japan in 1993 was 64.2 g, tens of times higher than that in Americans (7,8). Soybean is a rich source of the isoflavone genistein, which has been identified as a putative cancer chemopreventive agent (9). The daily intake of genistein and its [beta]-glucoside conjugated form, genistin, in the Japanese is estimated to be 1.5-4.1 and 6.3-8.3 mg, respectively, from the intake of soy products (10). In contrast, the daily consumption of total isoflavones by the British is calculated to be <1 mg (5). Adlercreutz et al. (4) also reported plasma concentrations of isoflavones in Japanese males to be 7-110 times higher than in Finnish males. Urinary excretion of genistein is 40 times higher in the Japanese than in Caucasian populations (6,11). Hence the daily intake of genistein is significantly higher in Japanese than Western European or American people (3).

Genistein is a protein tyrosine kinase inhibitor and it also inhibits DNA topoisomerases and other critical enzymes involved in signal transduction (11-13). In addition, antioxidant effects (14) and inhibition of angiogenesis have been reported (11). Its chemical structure shows a relationship to estrogenic steroids and genistein possesses weak estrogenic activity and has been shown to act in animal models as an anti-estrogen (5,15-17). It stimulates sex hormone-binding globulin (SHBG) production, which may lower the risk of hormone-related cancers by decreasing the amount of free and active hormones in the blood (5,18). Genistein has been demonstrated to suppress mammary cancer development in rats (19,20).

Although possible anti-carcinogenic properties of genistein have been suggested, reports concerning direct effects against prostate cancer cells and mechanisms of action are limited (21). In the present study, two abundant soybean isoflavones (genistein and daidzein) and their glucosides (genistin and daidzin) were tested to compare their effectiveness at cell growth inhibition of the LNCaP human prostatic cancer cell line. Induction of apoptosis and suppression of prostate-specific antigen (PSA) expression by genistein were also examined.

MATERIALS AND METHODS

Chemicals

Genistein, genistin and daidzin were purchased from Wako Pure Chemical Industries (Osaka, Japan). Daidzein was purchased from Extrasynthèse (Genay, France). The structures of the soybean isoflavones and their [beta]-glucoside conjugates used in the present study are shown in Fig. 1.


Figure 1. Structures of soybean isoflavones and their glucosides. Glc = glucose.

Cell Culture

The human prostatic cancer cell line LNCaP was obtained from the American Type Culture Collection (Rockville, MD) and cultured in RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin G and 100 µg/ml streptomycin at 37°C in 5% CO2.

Measurement of Proliferation Rates

LNCaP cells were seeded at 4 × 103 cells per well in 96-well microplates and allowed to attach for 24 h, then each isoflavone was added to the medium at various concentrations. After 72 h, cell growth was assessed as follows, using a WST-1 assay (Wako Pure Chemical Industries). Briefly, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) and 1-methoxy-5-methylphenazinium methylsulfate (1-methoxy PMS) (Wako Pure Chemical Industries) were added to the wells and incubated for 2 h, then cell growth was assessed by measuring the absorbance at 450 nm. Six replicated wells were used for each experimental condition.

Measurement of DNA Synthesis

DNA synthesis was evaluated using a kit from Boehringer Mannheim (Mannheim, Germany) as follows. LNCaP cells were plated and exposed to genistein as described above. After 2 h, 5-bromo-2-deoxyuridine (BrdU) was added to the medium to give a final concentration of 10 µM. After 2 h of additional incubation, culture medium was removed and BrdU incorporation was measured by an enzyme-linked immunosorbent assay (ELISA).

Nuclear Morphology

Cells were plated in culture plates at a concentration of 5 × 104 cells/ml. On the next day, genistein was added to the medium at a final concentration of 150 µM and culturing was continued for 24 h. Then both attached and detached cells were collected, fixed in 4% paraformaldehyde, stained with 0.17 mM Hoechst 33258 and examined under a fluorescent microscope.

DNA Fragmentation Assay

Cells were plated and treated with genistein as described above. After 24 h, both attached and detached cells were collected and lysed in 10 mM Tris-HCl (pH 7.4), 10 mM EDTA and 0.5% Triton X-100 for 10 min on ice. The lysates were centrifuged at 18 000 g for 20 min and the supernatants were incubated with 0.4 mg/ml RNase A (Worthington Biochemical, Freehold, NJ) for 1 h at 37°C, then with 0.4 mg/ml proteinase K (Merck, Darmstadt, Germany) for 1 h at 37°C. The DNA was precipitated at -20°C by adding an equal volume of propan-2-ol and 0.5 M NaCl, loaded on to 2% agarose gels containing 0.5 µg/ml ethidium bromide and electrophoresed in Tris-borate-EDTA (TBE) buffer. Gels were photographed under UV light.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblot Analysis

Cultured cells were placed in SDS-sample buffer (62.5 mM Tris-HCl, pH 6.8, 5% [beta]-mercaptoethanol, 2% SDS), sonicated for 10 s and then heated at 100°C for 5 min. The protein lysates were separated on 12% SDS-PAGE gels and transferred electrophoretically on to poly(vinylidene difluoride) (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA). After blocking with 2% non-fat skim milk in TTBS (20 mM Tris-HCl, pH 7.5, 140 mM NaCl, 0.05% Tween 20), the membranes were incubated with a 1:2000 dilution of anti-PSA polyclonal antibody (Dako, Glostrup, Denmark) in TTBS containing 1% bovine serum albumin. After washing with TTBS, they were incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-rabbit immunoglobulin (Amersham, UK), washed and developed using an enhanced chemiluminescence (ECL) system (Amersham). As an internal control, actin levels were also determined using anti-actin polyclonal antibody (Biomedical Technologies, Stoughton, MA).

RESULTS

LNCaP cells were cultured for 3 days in the presence of each soybean isoflavone at various concentrations and then viable cells were evaluated by WST-1 assay (Fig. 2). Among four isoflavones tested, genistein inhibited the growth of LNCaP most effectively, with an IC50 value of 40 µM. Daidzein showed a weak inhibitory effect on the growth of LNCaP. The [beta]-glucoside conjugates genistin and daidzin exerted far less influence than their aglycones genistein and daidzein, respectively. Treatment with genistein also reduced BrdU incorporation. The amounts of BrdU taken up by LNCaP between 2 and 4 h after the exposure to 37.5, 75 and 150 µM genistein were 74, 65 and 47% of control cells, respectively, consistent with the effects on cell growth.


Figure 2. Effect of isoflavones and their glucosides on the growth of the LNCaP human prostatic cancer cells. Cells were cultured in the presence of various concentrations of genistein ([open circle]), daidzein ([open square]), genistin ([solid circle]) and daidzin ([solid square]) After culturing for 3 days, cell viability was examined by a WST-1 assay. Cell viability is expressed as a percentage of the control cell value. Each point represents mean ± SE data for six wells.

When LNCaP cells were treated as monolayer sheets with genistein at concentrations >75 µM, they began to detach to some extent after 12 h and floating cells increased time dependently. Fluorescent microscope examination revealed these floating cells to show characteristic nuclear features of apoptosis such as chromatin condensation and nuclear fragmentation (Fig. 3). DNA was extracted from LNCaP cells cultured in the absence or presence of 75 or 150 µM genistein for 24 h, then determined using agarose gel electrophoresis. Fragmented DNA was observed in the cells treated with 75 or 150 µM genistein, but not in the cells without genistein treatment (Fig. 4). A DNA ladder pattern was clearly observed for LNCaP cells treated with 150 µM genistein (Fig. 4). The DNA fragments consisted of multimers of 180-190 base pairs, consistent with internucleosomal cleavage of chromatin DNA by an endonuclease.


Figure 3. Nuclear morphology of LNCaP cells treated with 150 µM genistein for 24 h. After the treatment, cells were stained with Hoechst 33258 and examined under a fluorescent microscope.


Figure 4. Effect of genistein on DNA fragmentation of LNCaP cells. Cells were cultured in the absence (lane 1) or presence of 75 µM (lane 2) or 150 µM (lane 3) genistein. After 24 h, fragmented DNA was extracted from LNCaP cells in lysis buffer and loaded on to a 2% agarose gel.

Analysis by immunoblotting showed that the cellular amounts of PSA in LNCaP cells were reduced when cells were treated with 15 µM genistein, at which dose cell growth was hardly affected, but the levels were not altered with 1.5 µM genistein, as shown in Fig. 5. A further decrease in PSA level was observed when cells were treated with 150 µM genistein. Densitometric analysis revealed that the amounts of PSA adjusted for the actin levels in the cells treated with 15 and 150 µM genistein for 24 h were 46 and 7%, respectively, of the control cell value.


Figure 5. Immunoblot analysis demonstrating the effects of genistein on the cellular amounts of PSA. LNCaP cells were treated with genistein at the indicated concentrations for 24 h. Cell lysates were subjected to immunoblot analysis, probed with anti-PSA (top) or anti-actin (bottom) antibodies.

DISCUSSION

In the present study, genistein was shown to be the most effective among the four soybean isoflavones tested in inhibition of the growth of human prostate cancer LNCaP cells. The estimated IC50 value, 40 µM, of genistein was comparable to that reported previously (21). Both suppression of DNA synthesis and induction of apoptosis were observed on treatment of the cells with genistein. These effects of genistein are not specific to LNCaP; cell growth inhibition and induction of apoptosis were also observed in another human prostate cancer cell line, DU145 (data not shown). Cellular amounts of PSA, a glycoprotein produced in the prostate having chymotrypsin-like serine protease activity and a tumor marker for prostate cancer (22), were also decreased dose dependently by genistein. The suppression of PSA expression was observed at a concentration of 15 µM, with which no growth inhibition of the cells was evident. This may be related to the effects of genistein on protein synthesis and/or signal transduction for PSA expression. Daidzein exhibited a weak inhibitory effect on the growth of LNCaP cells. Genistin and daidzin, the [beta]-glucoside forms of genistein and daidzein, respectively, showed much less growth inhibition activity than their aglycones. Genistein can be absorbed in the upper small intestine (23,24), whereas the [beta]-glucoside conjugate, genistin, needs conversion to an aglycone through the action of a [beta]-glucosidase produced by the intestinal bacteria before being taken up (5,23). Therefore, genistin may show anti-carcinogenetic activity after absorption from the intestine in vivo.

The present results support the view that genistein is a candidate component in soybeans accounting for the lower risk of prostate cancer in the Japanese. However, the contribution of other components to the preventive effects of soy-based diets in prostate cancer should be investigated further, because soybeans contain several other anti-carcinogens such as protease inhibitors, phytosterols, saponins and inositol phosphates (9,25). It is also possible that some other chemopreventive substances could act additively or synergistically with genistein. Although a 6 µM plasma level of genistein has been detected in subjects after the consumption of soymilk (24), few reports are available concerning the possible in vivo toxicity at higher concentrations.

It should be clarified whether the inhibition of DNA synthesis and the induction of apoptosis in prostate cancer cells are really observed in vivo without inducing any adverse effects. As far as colon cancer is concerned, a recent report indicated that genistein enhanced azoxymethane-induced colon carcinogenesis in a rat model (26). Further animal experiments and intervention studies are needed to determine the in vivo chemopreventive potential of genistein for prostate cancer.

Acknowledgments

This study was supported by a grant from the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, R&G Promotion and Product Review of Japan and a Grant-in-Aid from the Ministry of Health and Welfare for the Second-Term Comprehensive 10-Year Strategy for Cancer Control. M. Onozawa is the recipient of a Research Resident Fellowship from the Foundation for Promotion of Cancer Research.

References

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10. Fukutake M, Takahashi M, Ishida K, Kawamura H, Sugimura T, Wakabayashi K. Quantification of genistein and genistin in soybeans and soybean products. Food Chem Toxicol 1996;34:457-61. MEDLINE Abstract

11. Fotsis T, Pepper M, Adlercreutz H, Hase T, Montesano R, Schweigerer L. Genistein, a dietary ingested isoflavonoid, inhibits cell proliferation and in vitro angiogenesis. J Nutr 1995;125:790S-7S. MEDLINE Abstract

12. Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, et al. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 1987;262:5592-5. MEDLINE Abstract

13. Markovits J, Linassier C, Fossé P, Couprie J, Pierre J, Jacquemin-Sablon A, et al.Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase II. Cancer Res 1989;49:5111-17. MEDLINE Abstract

14. Wei H, Wei L, Frenkel K, Bowen R, Barnes S. Inhibition of tumor promoter-induced hydrogen peroxide formation in vitro and in vivo by genistein. Nutr Cancer 1993;20:1-12. MEDLINE Abstract

15. Folman Y, Pope GS. The interaction in the immature mouse of potent oestrogens with coumestrol, genistein and other utero-vaginotrophic compounds of low potency. J Endocrinol 1966;34:215-25. MEDLINE Abstract

16. Folman Y, Pope GS. Effect of norethisterone acetate, dimethylstilboestrol, genistein and coumestrol on uptake of [3H]oestradiol by uterus, vagina and skeletal muscle of immature mice. J Endocrinol 1969;44:213-8. MEDLINE Abstract

17. Setchell KDR, Borriello SP, Hulme P, Kirk DN, Axelson M. Nonsteroidal estrogens of dietary origin: possible roles in hormone-dependent disease. Am J Clin Nutr 1984;40:569-78.

18. Mousavi Y, Adlercreutz H. Genistein is an effective stimulator of sex hormone-binding globulin production in hepatocarcinoma human liver cancer cells and suppresses proliferation of these cells in culture. Steroids 1993;58:301-4. MEDLINE Abstract

19. Lamartiniere CA, Moore JB, Brown NM, Thompson R, Hardin MJ, Barnes S. Genistein suppresses mammary cancer in rats. Carcinogenesis 1995;16:2833-40. MEDLINE Abstract

20. Murrill WB, Brown NM, Zhang J-X, Manzolillo PA, Barnes S, Lamartiniere CA. Prepubertal genistein exposure suppresses mammary cancer and enhances gland differentiation in rats. Carcinogenesis 1996;17:1451-7. MEDLINE Abstract

21. Peterson G, Barnes S. Genistein and biochanin A inhibit the growth of human prostate cancer cells but not epidermal growth factor receptor tyrosine autophosphorylation. Prostate 1993;22:335-45. MEDLINE Abstract

22. McCormack RT, Rittenhouse HG, Finlay JA, Sokoloff RL, Wang TJ, Wolfert RL, et al. Molecular forms of prostate-specific antigen and the human kallikrein gene family: a new era. Urology 1995;45:729-44. MEDLINE Abstract

23. Barnes S. Effect of genistein on in vitro and in vivo models of cancer. J Nutr 1995;125:777S-83S. MEDLINE Abstract

24. Xu X, Harris KS, Wang H-J, Murphy PA, Hendrich S. Bioavailability of soybean isoflavones depends upon gut microflora in women. J Nutr 1995;125:2307-15. MEDLINE Abstract

25. Kennedy AR. The evidence for soybean products as cancer preventive agents. J Nutr 1995;125:733S-43S. MEDLINE Abstract

26. Rao CV, Wang C-X, Simi B, Lubet R, Kelloff G, Steele V, et al. Enhancement of experimental colon cancer by genistein. Cancer Res 1997;57:3717-22. MEDLINE Abstract

Received February 12, 1998; accepted March 30, 1998
For reprints and all correspondence: Keiji Wakabayashi, Cancer Prevention Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan

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