ANTICANCER RESEARCH 28 : 2837-2842 (2008) Soy Extract Is More Potent than Genistein on Tumor Growth Inhibition HYEON-A KIM 1, KYU-SHIK JEONG 2 and YOO KYEONG KIM 3 1Department of Food and Nutrition, Mokpo National University; 2Veterinary Medicine and 3Department of Home Economics Education, Kyungpook National University, Daegu, South Korea Abstract. Soybean and soy products have received much in Asian countries compared with Western countries (1, 2). attention for their potential heath benefits. Recently it has The effects appeared to be dietary and not genetic since the been reported that the bioactivity of soy products is protection against breast cancer conferred on Asian women influenced by the degree of soy processing. This study was was lost upon emigration and exposure to Western lifestyles conducted to evaluate and compare the influence of diets within a few generations (3). The major difference between containing genistein and soy extract on the growth of the Asian and Western diets lies in soy consumption. The Asian estrogen-independent human breast cancer cells, MDA-MB- diet is characterized by a high content of soy and soy 231, implanted into female Balb/c mice. Four-week-old products (2, 4). Much effort has been made to identify the female athymic nude mice (Balb/c) were acclimatized to an bioactive components in soybeans which have the potential AIN-93G control diet for one week prior to initiating the to prevent breast cancer. experimental diets. The animals were placed into three Soybeans contain a number of biologically active treatment groups, each of which was provided with compounds that may be associated with chemoprevention containing DMSO, genistein (750 μg/g AIN-93G diet) or including isoflavones, protease inhibitors, inositol 0.6% soy extract (containing genistein at 750 μg/g AIN-93G hexaphosphate, lignans, phytosterols and saponins. diet) for three weeks from one week prior to the injection of Isoflavones have been of interest and thoroughly studied due MDA-MB-231 cells (1×10 6/site) and subsequently fed on the to their putative activities in preventing and reducing breast AIN-93G control diet until sacrifice. The tumor volumes cancer risk. Isoflavones are a group of flavonoids which are increased steeply in the control group and the genistein- similar to endogenous 17β-estradiol in structure and treated group. However, tumor growth was significantly biological activity (5, 6). Soybeans are the most common reduced in the soy extract-treated group compared to the source of isoflavones in human food. The major isoflavones control and genistein-treated groups. Immunohistochemistry found in soy are genistein, daidgein and glycitein which exist of proliferating cell nuclear antigen (PCNA) also revealed naturally in several glycoside forms and are transformed to that the soy extract treatment effectively reduced cell the active aglycone form by enzymatic reaction. Among proliferation of the implanted tumors. In conclusion, soy them, genistein, the predominant isoflavone of soybeans, has extract is more potent than genistein in the inhibition of been the focus of attention associated with its tumor growth, presumably resulting from the synergistic chemopreventi ve activity against breast cancer (5-7). effect of the various bioactive components in the soy extract. Several mechanisms have been proposed for the anticarcinogenic activity of isoflavones. Isoflavones have been Epidemiological studies have indicated that diet might be reported to inhibit the growth of various cancer cells through the one factor that explains the lower incidence of breast cancer modulation of genes related to cell growth and programmed cell death (6, 7). Genistein has been found to inhibit the activation of the nuclear transcription factor, NF-kappaB and the Akt signaling pathway, both of which are known to maintain a Correspondence to: Yoo Kyeong Kim, Department of Home balance between cell survival and apoptosis (8, 9). Genistein also Economics Education, Kyungpook National University, 702- targets the signaling pathway mediated by estrogen and androgen 701#1370 Sangyeok-dong, Buk-gu, Daegu, South Korea. Tel: +82 in the processes of carcinogenesis. Moreover, genistein is known 539505929, Fax: +82 539505924, e-mail: [email protected] to be an inhibitor of angiogenesis and metastasis (10, 11). Key Words: Genistein, soy extract, MDA-MB-231 cell, nude mice, However, genistein treatment has led to paradoxical actions on tumor growth. breast cancer cells depending on the concentration to which the 0250-7005/2008 $2.00+.40 2837 ANTICANCER RESEARCH 28 : 2837-2842 (2008) cells are exposed. Physiological concentrations (1 nM to 10 μM) Cell culture. The estrogen-independent human breast cancer cells, of genistein stimulated the growth of estrogen-dependent human MDA-MB-231, were routinely cultured in RPMI-1640 medium breast cancer (MCF-7) cells in vitro and in vivo through the (MEM; Sigma) supplemented with 15% fetal bovine serum (FBS; Atlanta Biologics, Norcross, GA, USA), 2 mM L-glutamine and estrogen receptor-α (12-16), whereas a high concentration of 80 μg/ml penicillin and grown at 37˚C with 5% CO 2. The cells genistein (>10 μM) inhibited the growth and survival of human were quantified by hemocytometer counting and were more than breast cancer cells via a route independent of estrogen receptors 90% viable, as determined by trypan blue exclusion. (12, 13, 16). Animal studies have demonstrated that the timing of exposure to genistein is also important in the development of Animal studies. Four-week-old female athymic nude mice (Balb/c) breast cancer (17-22). Exposure to dietary genistein from the were purchased from Harlan Laboratories (Indianapolis, IN, USA). neonatal through prepubertal periods protected against The animals were housed in a climate-controlled room (22±2˚C, 50±10% relative humidity) with a 12-hour light/dark cycle and chemically induced mammary cancer in rats. The protective provided with diet and water ad libitum . The mice were effects included increased latency, reduced tumor incidence and acclimatized to an AIN-93G control diet for one week prior to multiplicity, and more rapid maturation of undifferentiated end initiating the experimental diets. The animals were then randomly buds to differentiated lobules. In contrast, limiting exposure to assigned to one of three treatment groups. The treatment groups dietary genistein to the prenatal or adult periods did not were: DMSO (G1); genistein (750 μg/g AIN-93G diet) (G2) and ® predispose or protect against mammary cancer (22). In studies 0.6% Novasoy (containing genistein at 750 μg/g AIN-93G diet) using xenograft animal models, treatment with genistein before (G3). The animals were provided with one of the experimental diets for three weeks from one week prior to the injection of MDA-MB- tumor formation was effective for tumor suppression, however 231 cells (1×10 6/site) and then fed on AIN-93G control diet until genistein treatment after tumor formation stimulated tumor sacrifice. The MDA-MB-231 cells (1×10 6/site) were injected into development (17, 18, 20). both the right and left flank on the back of each animal. During the Soy isoflavones have received much attention for their study, tumor growth and body weight were monitored weekly and potential health benefits. In Asia, soybeans are consumed as dietary intakes were also measured every week. The animals were minimally processed foods and not as processed and purified sacrificed 13 weeks after cell inoculation and the tumors were ingredients, whereas in Western countries soy products are dissected for further examination. produced and consumed in highly processed forms such as Immunohistochemistry. Tumor cell proliferation was measured by soy protein isolates (80-90% protein) and isoflavone- immunohistochemical analysis using proliferating cell nuclear enriched products (40-70% isoflavones). These products may antigen (PCNA). The tumors were removed and fixed in 10% not have the same health benefits as the soy foods consumed neutral-buffered formalin and embedded in paraffin. Four-μm serial in Asian countries because they may have lost some of the ir sections were cut from the paraffin blocks and placed on slides. The bioactive components. In fact, previous research has shown slide sections were incubated in xylene to remove the paraffin and that a highly refined isoflavone-rich diet may have rehydrated using a series of graded alcohol. Visual staining was achieved using a mouse monoclonal PCNA antibody (Santa Cruz detrimental effects. Allred et al. (23, 24) reported that Biotechnology, Santa Cruz, CA, USA) followed by streptividine- isoflavone-supplemented diets containing an equal amount of horseradish peroxidase (HRP) conjugate and diaminobenzidine genistein differentially stimulated mammary tumor growth in (DAB) substrate. Both the PCNA-positive proliferating cells and the athymic mice based on the degree of processing. The diets total tumor cells were counted in three non-necreotic areas of each containing the more processed products showed more section using light microscopy at 400-fold magnification. stimulation of estrogen-dependent breast tumor growth. In the present study, the effects of dietary genistein and Statistical analysis. All the data are presented as the mean±standard error. Statistically significant differences ( p< 0.05) among the means soy extract containing equivalent genistein content s on were determined by one-way ANOVA using a PC-based version of mammary tumor growth in an estrogen-independent