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Biochemical and Molecular Actions of Nutrients

Genistein at a Concentration Present in Soy Infant Formula Inhibits Caco-2BBe Cell Proliferation by Causing G2/M Cell Cycle Arrest¹

Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801

³To whom correspondence should be addressed. E-mail: sdonovan{at}uiuc.edu.

	ABSTRACT

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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 µmol/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.

KEY WORDS: • soy isoflavone • intestine • phytoestrogen

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 ß 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.

The objective of this study was to investigate how intestinal cell dynamics are impacted by genistein at concentrations found in soy-based infant formula and at a lower concentration. Our hypothesis was that a low dose of genistein would stimulate intestinal cell proliferation and that genistein at the concentrations present in soy infant formula would inhibit intestinal cell proliferation. The Caco-2 brush border expressing (Caco-2BBe; American Type Culture Collection) human colon adenocarcinoma cell line that spontaneously differentiates into enterocyte-like cells was used as a model to investigate the effect of genistein on intestinal cell dynamics. These cells assemble a brush border and express the disaccharidases lactase and sucrase-isomaltase (22). Ultrastructurally, the brush border of the Caco-2BBe is similar to that in the small intestine of humans (23). Additionally, Caco-2 cells efficiently take up and accumulate genistein within the cell, and to some extent genistein is glucuronidated (24,25), similar to the uptake of genistein by the intestine in vivo.

	METHODS

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    Cell culture. Caco2-BBe (passages 56–61) was seeded into collagen-coated T-75 flasks and 24-well plates (Becton-Dickinson) at a density of 3.5 x 10⁴ cells/cm². Separate flasks or plates were seeded for each assay. The cells were cultured in DMEM (GibcoBRL) + 10% fetal bovine serum (FBS; GibcoBRL) at 37°C in 5% CO₂ to promote attachment. After 6 h, cells were washed with HBSS and switched to low serum medium (DMEM + 0.5% FBS) and pretreated with 5 µg/L of epidermal growth factor (Sigma) for 18 h to synchronize the cells. Subsequently, the cells were exposed to genistein (Sigma), which was dissolved in dimethyl sulfoxide (DMSO); DMSO was present at 0.01–0.3% in the medium. The cells were exposed to an increasing concentration of 0, 2, 3.7, 26, 52, and 111 µmol/L (0, 0.5, 1, 7, 14, and 30 mg/L) genistein to assess the impact of a range of genistein concentrations on intestinal cell proliferation. Additional experiments only used genistein treatments of 3.7 µmol/L (1 mg/L) as the low genistein dose and 111 µmol/L (30 mg/L) as the high genistein dose. Low serum medium + DMSO served as the control. The cells were exposed to the treatments for 24 or 48 h.

    Cell proliferation. After the 24-h genistein treatment period, 37 kBq (1 µCi) [³H]thymidine (Amersham) was added to the medium of each well of the 24-well plate and incubated for 3 h at 37°C. Subsequently, the media were removed to eliminate any unincorporated [³H]thymidine and the cell monolayer was washed and processed as described by Chao and Donovan (26). Incorporation of [³H]thymidine by the cells was determined in a liquid scintillation counter (Beckman LS 9000).

To assess whether genistein mediates Caco2-BBe cell proliferation through the estrogen receptor, the cells were treated with increasing concentrations of genistein in the presence or absence of 1 µmol/L ICI 182,780 (Tocris), an ER antagonist. The cells were exposed to the treatments for 24 h and then proliferation was measured as described above.

    Cell number. First, the conditioned medium was saved to collect detached cells floating in the medium, and then the cell monolayer was washed with HBSS. The cell monolayer was removed after 5–10 min of incubation with trypsin solution (Sigma) at 37°C. After the conditioned medium was readded, the cells were resuspended into a single-cell suspension and an aliquot was taken for cell counting. An equal volume of trypan blue solution 0.4% (Sigma) was added and cell numbers were counted on a hemacytometer. Cells dyed blue were considered nonviable.

    Apoptosis. Caspase-3 is a protease key in the execution of apoptosis and an early marker of apoptosis (27,28). Apoptosis was assessed using a caspase-3 colorimetric assay kit (R&D Systems). The single-cell suspension was collected by centrifugation at 100 x g (Beckman GS-6R Centrifuge) and the assay was conducted following the manufacturer’s instructions. Briefly, the cells were lysed and the lysates were transferred to wells in a 96-well flat-bottom microplate. A peptide with the caspase-3 target motif DEVD (29) bound to the chromophore p-nitroanilide was added and incubated at 37°C for 1–2 h. The intensity of the developed color was read at 405 nm in a microplate reader (SpectraMax Plus; Molecular Devices). The results are expressed as a percentage of the control.

    Cell cycle. After 24 and 48 h exposure to treatment, the single-cell suspension was pelleted at 100 x g (Beckman GS-6R Centrifuge), washed with cold PBS, and then resuspended to 2 x 10⁶ cells/mL. One milliliter was transferred to a polypropylene tube to which 3 mL of absolute ethanol was added. The cells were fixed in this solution and stored at –20°C for later analysis. The fixed cells were pelleted at 250 x g and washed with cold PBS. A total of 100 µL of 200 mg/L RNase A (Ambion) was added to the cells and incubated in a water bath at 37°C for 30 min. Thereafter, 1.2 mL of propidium iodide (PI; Sigma) staining solution (50 mg/L PI in 3.8 mmol/L sodium citrate) was added and incubated for 20 min at room temperature in the dark. The cells were analyzed in a flow cytometer (Coulter XL-MCL).

    Western immunoblot. After 24 and 48 h exposure to treatment, cells were lysed and the protein concentrations of the cell lysates were determined by a modified Lowry assay (30). Proteins were separated by SDS-PAGE on a 10% Tris-HCl gel (Bio-Rad) and electrotransferred to nitrocellulose membranes. Antibodies against cyclin B1 (Santa Cruz Biotechnology), cyclin D (Upstate Biotechnology), and actin (Santa Cruz Biotechnology) were used to probe the separate membranes. Membranes were probed with 2–8 µg of the antibody diluted in Tris-buffered saline with Tween 20 + 1% bovine serum albumin at 4°C overnight. The next day, membranes were incubated with goat anti-rabbit horseradish-peroxidase–conjugated antibody and detected colorimetrically by Bio-Rad’s Opti-4CN kit. Bands were quantified by densitometry (Image Station 440CF; Kodak). Cyclin B1 and cylin D protein band densities were standardized by actin.

    Statistics. Computations were performed using the general linear model procedure and post hoc LSD test with the Tukey-Kramer test to adjust for multiple comparisons. For the experiment investigating proliferation at different genistein concentrations in the presence and absence of ICI, a two-way ANOVA was done with the LSD post hoc test. Statistical determinations were computed using SAS Version 8e (SAS Institute). Statistical significance was set at P 0.05.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Proliferation. Genistein exerted a biphasic effect on intestinal cell proliferation (Fig. 1). Compared to control, thymidine incorporation was increased 28% in the intestinal cells exposed to 3.7 µmol/L genistein (P = 0.001). In contrast, genistein concentrations of 26–111 µmol/L reduced thymidine incorporation by 67–94% (P < 0.0001), which was different compared to 0, 2, and 3.7 µmol/L genistein. The presence of the ER antagonist ICI 182,780 negated the increase in proliferation induced by the low doses (2 and 3.7 µmol/L) of genistein. However, at the high doses of 52 and 111 µmol/L the addition of ICI did not lower the already suppressed thymidine incorporation (Fig. 2). The addition of ICI 182,780 to the control no-genistein group also resulted in a reduction in proliferation compared to proliferation in the absence of ICI. However, this can be attributed to the pH indicator, phenol red, in the cell culture medium. Impurities present in phenol red preparations have been shown to have estrogenic activity (31,32). To verify this, the same proliferation experiment was repeated in phenol-red–free medium. The experiment demonstrated similar effects of genistein on cell proliferation and no reduction in proliferation in the control group in the presence of ICI (data not shown).

View larger version (12K): [in this window] [in a new window]   FIGURE 1 [3H]Thymidine incorporation of Caco-2BBe cells exposed to 0, 2, 3.7, 26, 52, and 111 µmol/L genistein for 24 h. Values are means ± SD, n = 4–10. Means without a common letter differ, P 0.05.

View larger version (17K): [in this window] [in a new window]   FIGURE 2 [3H]Thymidine incorporation of Caco-2BBe cells exposed to 0, 2, 3.7, 26, 52, and 111 µmol/L genistein in the absence and presence of 1 µmol/L ICI 182,780 for 24 h. Values are means ± SD, n = 8. Means without a common letter differ, P 0.05. Proliferation stimulated through the estrogen receptor (as indicated by a reduction in proliferation with the addition of ICI) in the absence of genistein in the control group is attributable to phenol red in the culture medium.

View larger version (15K): [in this window] [in a new window]   FIGURE 3 Cell count of Caco-2BBe cells exposed to 0, 3.7, and 111 µmol/L genistein for 24 h. Cells were stained with trypan blue and cells dyed blue were considered nonviable. Values are means ± SD, n = 8. Means without a common letter differ, P 0.05. There was no difference in the percentage of nonviable cells.

View larger version (12K): [in this window] [in a new window]   FIGURE 4 Effects of a 24-h exposure to 0, 3.7, and 111 µmol/L genistein on caspase-3 activity in Caco-2BBe cells. Apoptosis was assessed by a colorimetric caspase-3 activity assay. Values (percentage of control) are means ± SD, n = 4. Means without a common letter differ, P 0.05.

View larger version (39K): [in this window] [in a new window]   FIGURE 5 Effects of 24- and 48-h exposure to 0, 3.7, and 111 µmol/L genistein on the cell cycle of Caco-2BBe cells. Values are the percentage of the cell population in the G0/G1 and G2/M phase of the cell cycle, n = 2.

View larger version (21K): [in this window] [in a new window]   FIGURE 6 Effects of 24- and 48-h exposure to 0, 3.7, and 111 µmol/L genistein on cyclin B1 (A) and cyclin D (B) protein abundance in Caco-2BBe cells. Cyclin B1 and cyclin D protein levels were normalized by actin protein levels. Values are means ± SD, n = 3. Means without a common letter differ, P 0.05.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION LITERATURE CITED

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.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 2002, April 2002, New Orleans, LA [Chen, A.-C. & Donovan, S. M. (2002) Genistein at a concentration present in soy infant formula inhibits intestinal cell proliferation by causing G2/M cell cycle arrest. FASEB J. 16: A281 (abs.)] and Digestive Disease Week 2002, May 2002, San Francisco, CA [Chen, A.-C. & Donovan, S. M. (2002) Genistein mediates intestinal cell proliferation through the estrogen receptor. Gastroenterology 122: A372 (abs.)].

² An-Chian Chen was 1 of 12 finalists in the 2002 ASNS Proctor & Gamble Graduate Student Research Awards abstract competition.

⁴ Abbreviations used: Caco-2BBe, Caco-2 brush border expressing cell line; Cdk, cyclin-dependent kinase; DMSO, dimethyl sulfoxide; ER, estrogen receptor; FBS, fetal bovine serum; LPH, lactase phlorizin hydrolase; MAP, mitogen-activated protein; PI, propidium iodide.

Manuscript received 17 November 2003. Initial review completed 15 December 2003. Revision accepted 18 February 2004.

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