J Nutr. 2004 Jun;134(6):1303-8.
Genistein at a concentration present in soy infant formula inhibits Caco-2BBe cell proliferation by causing G2/M cell cycle arrest.
Chen AC, Donovan SM.
Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
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Fifteen percent of all U.S. infants are fed soy formulas containing up to 47 mg/L of isoflavones (>65% as genistin + genistein); thus, these infants' intestines are exposed to a high dose of genistein, a phytoestrogen and tyrosine kinase inhibitor. Little attention has been focused on genistein's impact on the developing intestine. We hypothesized that a high dose of genistein would inhibit intestinal cell growth. Caco-2BBe human intestinal cells were exposed to 0, 3.7, and 111 micro mol/L (0, 1, and 30 mg/L) genistein in DMEM + 0.5% fetal bovine serum for 24-48 h. Cell number, thymidine incorporation, apoptosis, and cell cycle analyses were performed. The low genistein concentration increased intestinal cell proliferation by 28% (P = 0.001), but did not affect cell number or caspase-3 activity compared to the control. Furthermore, the addition of ICI, an estrogen receptor antagonist, negated the proliferative effect of the low genistein. In contrast, the high genistein concentration reduced cell number by 40%, proliferation by 94%, and caspase-3 activity by 50% compared to the control (P < 0.05). Cell cycle analysis after 48 h exposure to high genistein revealed 37% of cells in G0/G1 and 35% in G2/M vs. 71% in G0/G1 and 17% in G2/M for the control and low genistein groups. Thus, a biphasic effect of genistein was seen with a low dose stimulating intestinal cell proliferation through the estrogen receptor, whereas a high dose of genistein inhibited intestinal cell proliferation and altered cell cycle dynamics. A high dose of genistein may potentially compromise intestinal growth.

From the full text article:

Currently, 25% of formula-fed infants, or 15% of all infants, in the United States are fed soy-protein–based formulas (1). Soybeans contain a physiologically active class of phytoestrogens called isoflavones. The concentration of total isoflavones in soy infant formulas ranges from 32 to 47 mg/L (2), compared to 6 µg/L in human breast milk (2–4). Genistein, predominately present in the glycosidic conjugated form called genistin, comprises 65% of the total isoflavone content in soy formula (3). Based on the typical volume of formula consumption of 900–1000 mL/d, a 4-mo-old, soy-formula–fed infant consumes 28–47 mg (or 6–9 mg/kg body weight/d) of isoflavones each day. This dose is 6- to 11-fold higher than the dose found to have physiological effects in adult humans (5).

Because the intestine of the soy-formula–fed infant is exposed to high concentrations of genistein continuously and on a daily basis, it is important to consider how ingested genistein modulates neonatal intestinal development. A typical human infant has abundant lactase phlorizin hydrolase (LPH)4 in the small intestine to digest lactose present in breast milk and cow’s-milk–based infant formula (6). LPH can also hydrolyze genistin to genistein (7), and genistein is taken up by the enterocytes. Recent data indicate that genistein is taken up by the intestinal cells and is enriched in intestinal tissue during the first 30 min of administration (8–10). Although genistein is glucuronidated within the enterocyte, about 3% remains as free genistein within the enterocyte (10). Consequently, the biologically active genistein can have an effect on the enterocyte.

The intestine serves a number of important physiologic functions, including digestion, absorption, and barrier and immune function. Renewal of the intestinal villi occurs through a highly regimented progression of proliferation, differentiation, and apoptosis (11). Genistein has the potential to modulate intestinal cell dynamics through disrupting intracellular signaling pathways by inhibiting tyrosine kinases (12) or via direct interaction with estrogen receptors (ER) on intestinal cells (13–15). The gastrointestinal tract expresses mainly the beta form of the ER (14–16) and ERß has a high affinity for genistein (17). Estrogen, acting through the ER to activate protein tyrosine kinases, stimulates cell proliferation (18,19). Additionally, many growth factors that regulate intestinal growth and differentiation, such as insulin-like growth factor-I and epidermal growth factor, exert their physiological actions via receptor tyrosine kinases that initiate phosphorylation of intracellular proteins (20). However, genistein is a potent and specific inhibitor of tyrosine kinase activity (12) and can disrupt these intracellular signal transduction pathways. Thus, at low concentrations genistein, acting as a phytoestrogen, may stimulate intestinal development via intestinal ER. At high concentrations genistein, acting as an inhibitor of tyrosine kinase and topoisomerase II (21), can disrupt intracellular phosphorylation pathways and DNA replication, respectively, resulting in growth impairment.

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Discussion

Our results demonstrated a biphasic effect of genistein on the growth of cultured human intestinal cells. At a low concentration (3.7 µmol/L), genistein exerted a proliferative effect whereas genistein concentrations ranging from 26 to 111 µmol/L inhibited intestinal cell proliferation. The concentrations of genistein, including the contribution from genistin digestion to genistein, provided by soy infant formula fall in the proliferation inhibitory range tested. The additional experiments used the highest genistein concentration in the range (111 µmol/L) to elucidate mechanisms. Exposure to the low dose of genistein (3.7 µmol/L) stimulated cell proliferation. Although an increase in cell number was not observed, in a separate but similarly conducted experiment (data not shown) an increase in cell number (by 34%) was evident after 48 h, but not at 24 h. This lag was expected because there is a gap between DNA synthesis and actual mitosis when the cells divide and an increase in cell number would be detectable. A high dose of genistein modulated cell numbers through regulation of cell cycle, proliferation, and apoptosis. At 111 µmol/L genistein, proliferation and apoptosis were inhibited, and cells accumulated in the G2/M phase of the cell cycle. The arrest at G2/M prevented the cells from completing the cell cycle and proliferating. Collectively, these effects led to a drop in cell numbers. Although there was a decrease in apoptosis, the decrease in proliferation was greater, thus favoring a reduction in overall cell numbers.

We further investigated whether the high dose of genistein caused G2/M cell cycle arrest by affecting the protein abundance of key cell cycle regulatory proteins. Progression through the cell cycle is regulated by cyclin-dependent kinases (Cdks), a family of serine/threonine protein kinases that phosphorylate a variety of protein substrates that control the cell cycle and are activated by cyclins (33). The cellular concentrations of cyclins rise and fall with the stages of the cell cycle, whereas the levels of Cdks remain relatively stable, but must bind to the appropriate cyclin in order to be activated (33). Cyclin B1 is a cell cycle regulatory protein that is induced at the G2/M transition and drops off as the cell exits mitosis (33). The higher cyclin B1 protein level observed in the high genistein group could prevent the cells from exiting G2/M, thus corroborating the observed cell cycle arrest at G2/M and reduced cell proliferation in the high genistein group. Research in breast cancer cell lines has shown similar effects, with genistein blocking cell cycle progression at G2/M by increasing protein expression of cyclin B1 (34,35).

We also investigated the abundance of cyclin D because this protein regulates entry into and progression of the cell cycle at G1 (33). At 24 h, there was a decrease in cyclin D protein level in the high genistein group. The lower cyclin D level suggests that cells are not entering the cell cycle, which supports the decrease in cell proliferation observed in the high genistein group. However, we also observed decreased cyclin D protein abundance in the low genistein group, which counters the increased cell proliferation induced by a low dose of genistein. Previous work in mammary cells demonstrated that genistein at low concentrations induced the synthesis of cyclin D1 for progression through the cell cycle (18). Since we saw a decrease in cyclin D in genistein-stimulated proliferating intestinal cells, it is possible that genistein mediates other key G1 cell cycle regulators. For example, estrogen has been shown to regulate the abundance of CDK2, CDK4, p21 (waf1/cip1), and p27(kip1) to promote the progression of mammary cells through the cell cycle (36–38).

Previous studies also have shown that genistein influences intestinal cell dynamics. Consistent with our findings, in the IEC18 untransformed neonatal rat small intestine cell line, genistein concentrations greater than 1 mg/L (3.7 µmol/L) dramatically inhibited cell proliferation after a 5-d exposure (39). IEC-6 (rat fetal nontransformed intestinal cells) cell numbers were reduced to 5% of the control when exposed to 80 µmol/L genistein for 48 h (40). These data show that the inhibitory effect of genistein on cell growth is not limited to transformed cells. When exposed to 100 µmol/L genistein for 48 h, Caco-2 and HT-29 (both human colon adenocarcinomas) cell numbers were reduced 75 and 60%, respectively, compared to the control (40). Furthermore, in HT-29 cells, a 4-d exposure to 60 and 150 µmol/L genistein resulted in 54 and 94% apoptotic cells, respectively (41). However, in our study we observed a decrease rather than an increase in apoptosis in the high genistein group. In our study the Caco-2BBe cells were exposed to genistein for a short period (24 h). It has been demonstrated that within 48 h HT-29 cells had repaired initial DNA breakage induced by 10–30 µmol/L genistein (41). Likewise, perhaps the Caco-2BBe cells were able to revert early apoptotic effects; thus a 24-h exposure was insufficient to allow the apoptosis-inducing effects of genistein to be evident.

Low concentrations of genistein demonstrated proliferative effects in the current study and earlier work found in the literature. Doses of genistein at <3.7 µmol/L stimulated cell growth in the IEC18 cell line (39). Cell proliferation was noted in HT-29 cells treated with 1–2 µmol/L genistein (41). In ER-positive MCF-7 breast carcinoma cells, genistein doses of 1 nmol/L to 10 µmol/L stimulated growth (42). However, in ER-negative breast carcinoma cells, the same concentrations of genistein suppressed proliferation (42). Evidently, the estrogen receptor is necessary for genistein to stimulate cell growth, but is not required for genistein to inhibit cell growth. Interestingly, the amount of ER determined in subconfluent Caco-2 cells was similar to that in MCF-7 cells, and estradiol stimulated Caco-2 cell growth (19). Thus, the proliferative effects of genistein in ER+ MCF-7 and Caco-2 cells may function through similar mechanisms. Herein, the ER antagonist ICI 182,780 negated the increase in proliferation induced by low genistein concentrations, indicating that genistein stimulated proliferation in Caco-2BBe cells through the ER and that ER has a role in signaling for cellular proliferation. At the 2 highest genistein doses, the addition of ICI 182,780 did not further the reduction in proliferation, likely due to the fact that at high doses, genistein’s ability to inhibit tyrosine kinases supersedes that of its estrogenic activity. Estradiol-stimulated cell growth occurs through mitogen-activated protein (MAP) kinases and is dependent on activating the protein tyrosine kinase, c-src (19). Genistein in high doses inhibits tyrosine kinases and thus inhibits cell proliferation signaling through the ER. Consequently, the addition of an ER antagonist would not further reduce cell proliferation because the signaling pathway was halted downstream by the tyrosine kinase inhibitory actions of genistein. Indeed, it has been shown that genistein at 40 and 80 µmol/L inhibited estradiol stimulation of Caco-2 cell growth by inactivating c-src, thus leading to the inactivation of MAP kinases (19). Overall, genistein at low doses acts as an ER agonist to promote cell growth, but at higher concentrations the ability of genistein to inhibit tyrosine kinase activity supersedes its proliferative effects.

The intestine is a continuously renewing tissue that is constantly undergoing proliferation; thus, intestinal cells could be highly susceptible to the effects of genistein on cell cycle dynamics. Herein we report that exposure to genistein at the concentrations present in soy infant formula inhibited intestinal cell proliferation. The reduction in proliferation was due to cell cycle arrest at G2/M, which may be the result of a higher level of cyclin B1. Additionally, we demonstrated that genistein mediated intestinal cell proliferation through the estrogen receptor. The ability of genistein to stimulate and inhibit intestinal cell proliferation may have implications for soy formulas on intestinal growth.

Categories: 2004, Soy, Phytoestrogens, Endocrine, Estrogen, IGF-1, Gastrointestinal, Digestive malabsorption, Nutrition and diet