Galectin-3 mediates genistein-induced G2/M arrest and inhibits apoptosis
Huei-Min Lin1,4,
Bong-Ki Moon3,
Fei Yu1 and
Hyeong-Reh Choi Kim1,2,5
1 Department of Pathology and
2 Breast Cancer Program, Barbara Ann Karmanos Cancer Institute, Wayne State University, School of Medicine, Detroit, MI 48201, USA and
3 Department of Anesthesiology, Ajou University School of Medicine, Suwon, Korea
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Abstract
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Many recent studies have focused on potential chemopreventive activities of dietary genistein, a natural isoflavonoid compound found in soy products. Genistein has been implicated in anticancer activities, including differentiation, apoptosis, inhibition of cell growth and inhibition of angiogenesis. In previous studies, genistein was shown to induce apoptosis and cell cycle arrest at G2/M in several cancer cell lines in vitro, which is associated with induction of p21WAF1/CIP1, a universal inhibitor of cyclin-dependent kinases. At present, the molecular basis for diverse genistein-mediated cellular responses is largely unknown. In the present study, we investigated whether galectin-3, an anti-apoptotic gene product, regulates genistein-mediated cellular responses. We show that genistein effectively induces apoptosis without detectable cell cycle arrest in BT549, a human breast epithelial cell line which does not express galectin-3 at a detectable level. In galectin-3 transfected BT549 cells, genistein induced cell cycle arrest at the G2/M phase without apoptosis induction. Interestingly, genistein induces p21WAF1/CIP1 expression in galectin-3-expressing BT549 cells, but not in control BT549 cells undergoing apoptosis. Collectively, the results of the present study suggest that galectin-3, at least in part, is a critical determinant for genistein-mediated cell cycle arrest and apoptosis, and genistein induction of p21WAF1/CIP1 is associated with cell cycle arrest, but not required for apoptosis induction.
Abbreviations: Ac-DEVD-amc, acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl coumarin;; ER, estrogen receptor;; PBS, phosphate-buffered saline;; SDS, sodium dodecyl sulfate.
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Introduction
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Epidemiological studies suggest that the soybean isoflavonoid genistein (4,5,7-trihydroxyisoflavone phytoestrogen) may decrease the incidence of certain types of cancer and reduce the age-adjusted death rates from breast cancer (13). Consistently, genistein has been shown to mediate cell differentiation (4), inhibit angiogenesis (5,6) and be cytotoxic in a wide variety of cancer cell lines in vitro (4,710). Experiments in mice also provide evidence for genistein-mediated cytotoxic/cytostatic activity (11). Several possible mechanisms for genistein's anticancer activity have been suggested. These include inhibition of protein tyrosine kinase and topoisomerase (7,8,12,13). In contrast to the anticancer activity of genistein, genistein enhances carcinogen-induced tumor formation in the mouse colon (14). Similarly, maternal exposure to genistein dose-dependently increases carcinogen-induced mammary tumor progression in female rat offspring, mimicking the effects of estrogen exposure (15). These studies suggest that genistein may enhance tumorigenic potential in an organ-specific and/or developmentally regulated manner. At present, the molecular basis for genistein-mediated diverse cellular effects is not known.
Genistein induces apoptosis and cell cycle arrest at G2/M in a variety of cancer cell lines in vitro, which is associated with p21WAF1/CIP1 induction, a universal inhibitor of cyclin-dependent kinases (4,10,16). Efforts have been made to determine the gene products critical for mediating genistein-induced cellular responses. Since early studies showed that genistein binds estrogen receptors (ERs) (32,33), the role of ERs on genistein-mediated effects in cancer cells was examined. No relationship was found between ER expression and genistein-induced cell cycle arrest or apoptosis (7). Similarly, inhibition of cell growth by genistein was shown to be independent of the tumor suppressor gene product p53 (16).
In the present study, we investigated whether galectin-3, an anti-apoptotic gene product, modulates genistein-mediated cellular responses. Galectins are a family of proteins that bind to galactose-containing ligands (17). Galectin-3, a 30 kDa member of the galectin family, is widely found in epithelial and immune cells, and is highly expressed in various human tumor cells, including breast cancer (1822). Although the exact mechanism is unknown, galectin-3 expression is associated with neoplastic progression and metastatic potential (1825). A recent study showed that galectin-3 inhibits anti-Fas antibody and staurosporine-induced apoptosis in T-lymphocytes (26). In accordance with this, we have demonstrated that galectin-3 inhibits apoptosis induced by cisplatin (27) and loss of cell adhesion (anoikis) (28), suggesting that the oncogenic activity of galectin-3 may involve apoptosis inhibition. Here, we report that genistein arrests cells at the G2/M phase in galectin-3-expressing human breast epithelial cells without detectable apoptosis induction, while it rapidly induces apoptosis in the control cells without detectable cell cycle arrest. In addition, we provide evidence indicating that genistein-induced p21WAF1/CIP1 is associated with cell cycle arrest at G2/M but is not required for apoptosis.
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Materials and methods
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Cell culture and genistein treatment
The human breast cancer cell line BT549 was obtained from Dr E.W.Thompson, Vincent T.Lombardi Cancer Research Center, Georgetown University Medical Center, Washington, DC. Galectin-3-transfected BT549 cells (BT549-Galwt) were previously established by introducing an expression vector containing human galectin 3 cDNA into BT549 parental cells (27,28). BT549 cells transfected with the control vector conferring neomycin resistance are referred to as BT549neo. BT549 cells expressing a mutant galectin-3 in which Gly182 of the NWGR motif was replaced with Ala (27,28) are referred to as BT549-Galm. Cells were cultured using DMEM/F12 supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine and 0.5 µg/ml fungizone in 95% air/5% CO2 at 37°C. After cells had been seeded for 46 h, cells were treated with 45 or 90 µM genistein (Sigma, St Louis, MO).
Antibodies
An anti-human galectin-3 monoclonal antibody (mAb) was purchased from the American Type Culture Collection (Rockville, MD), anti-cyclin D1 mAb (Ab2) and anti-p21WAF1/CIP1 mAb from Oncogene Research (Cambridge, MA), anti-p27KIP1 polyclonal antibody from Santa Cruz Biotech (Santa Cruz, CA), and anti-human ß-actin mAb from Sigma.
Immunoblot analysis
Cell lysates were prepared using sodium dodecyl sulfate (SDS) lysis buffer (2% SDS, 125 mM TrisHCl pH 6.8, 20% glycerol). The lysates were boiled for 5 min and then clarified by a 20 min centrifugation at 4°C. Protein concentration was measured using BCA protein assay reagent (Pierce, Rockford, IL). Equal amounts of protein samples in SDS sample buffer (1% SDS, 62.5 mM TrisHCl pH 6.8, 10% glycerol, 5% ß-mercaptoethanol, 0.05% Bromophenol Blue) were boiled for 5 min and subjected to reducing SDSpolyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to a nitrocellulose membrane. The blot was blocked with 5% non-fat dry milk in 100 mM TrisHCl pH 7.5, 150 mM NaCl, 0.02% NaN3 and 0.2% Tween-20 (T-TBS) for 1 h at room temperature. The membranes were incubated with the appropriate primary antibody in 5% milk in T-TBS. After three washes with T-TBS, the blot was incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. The antigen was detected using the enhanced chemiluminescence detection system (Pierce) according to the manufacturer's instruction.
Determination of cell cycle distribution
Cells were trypsinized, washed with PBS and fixed with 70% ethanol. The fixed cells were spun down and resuspended in Hoechst staining solution at a concentration of 1 x 106 cells/ml and incubated for 3 min at room temperature. The Hoechst staining solution consisted of 3 mg/ml Hoechst 33258 (Sigma) in Tris buffer (2 mM MgCl2, 0.1% Triton X-100, 154 mM NaCl, 100 mM Tris, pH 7.5). The percentage of cells in each cell cycle phase was determined at the Imaging Flow Cytometry Core Facility at our institute.
Nuclear staining
Cells grown on coverslips were treated with 90 µM genistein. After 48 h of treatment, cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for
12 h at 4°C. The coverslips were removed from the tissue culture dish and cells were exposed to 1 µg/ml 2'-(4-hydroxyphenyl)-5-(4-methyl-1 piperazinyl)-2,5'-bi-1H-benzimidazole trihydrochloride pentahydrate (bisbenzimide, Hoechst 33258; Molecular Probes, Eugene, OR) in PBS for 15 min at room temperature, and washed with PBS. The cells were mounted in 0.1% phenylene diamine and 90% glycerol in PBS. Nuclear morphology was examined under UV illumination on a fluorescence microscope.
DEVDase activity assay
Cells were lysed in 50 mM Tris buffer (pH 7.5) containing 0.03% Nonidet and 1 mM dithiothreitol (DTT). Nuclei were removed by low-speed centrifugation (800 x g, 5 min), and the cytosol fraction was incubated with 40 µM acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl coumarin (Ac-DEVD-amc), 10 mM HEPES (pH 7.5), 50 mM NaCl and 2.5 mM DTT in a total volume of 200 µl for 60 min at 37°C. Fluoromethylcoumarin fluorescence, released by DEVDase (caspase) activity, was measured using 360 nm excitation. A CCD device (Instaspec IV; Oriel, Stratford, CT) fitted with a monochromator was used to measure the fluorescence emission spectrum. The intensity at the optimum wavelength (~460 nm) was measured. DEVDase activity was normalized per microgram of protein determined using a BCA protein assay kit (Pierce).
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Results
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Galectin-3 expression protects human breast epithelial cells against genistein-induced cytotoxicity
To examine the roles of galectin-3 on genistein-induced cytotoxic/cytostatic activity, control and galectin-3-overexpressing BT549 cells (BT549neo and BT549-Galwt, respectively) were treated with genistein and subsequently evaluated for their cell viability using the MTS cell proliferation assay (Promega, Madison, WI). These cells were chosen to study the roles of galectin-3 on genistein-mediated cellular effects, since the parental BT549 cells do not express galectin-3 at a detectable level. After exposure to 90 µM genistein for 24 h, ~80% of BT549neo cells remained viable, while the number of BT549-Galwt cells increased to ~180% (Figure 1A
). An additional 24 h exposure of cells to 90 µM genistein was cytostatic towards BT549-Galwt cells and was cytotoxic towards BT549neo cells (Figure 1B
). This shows that galectin-3 expression protects BT549 cells against genistein-induced cytotoxicity.

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Fig. 1. Galectin-3 protects BT549 cells from genistein-induced cytotoxicity. BT549neo, BT549-Galwt and BT549-Galm cells were treated with 45 or 90 µM genistein for 24 h (A) or with 90 µM genistein for 24 or 48 h (B). The percentage of cell survival was normalized to the respective control cells (no treatment). All experiments were performed in triplicate; the error bars represent the standard deviation.
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Structurally, galectin-3 is composed of two distinct domains: an N-terminal domain containing proline and glycine-rich sequences and a globular C-terminal domain containing the carbohydrate recognition site (18). Galectin-3 contains four amino acid residues, NWGR, that are conserved in the BH1 domain of the Bcl-2 family. This motif is critical for Bcl-2's anti-apoptotic activity (29). As in the Bcl-2 protein, substitution of the Gly182 residue with Ala in the NWGR motif of galectin-3 abrogates its anti-apoptotic function (27,28). To test whether the NWGR motif is required for galectin-3 inhibition of genistein-mediated cytotoxicity, we examined the effect of genistein on BT549-Galm cells, BT549 cells expressing mutant galectin-3 in which the Gly182 of the NWGR motif was substituted with Ala (27,28). As shown in Figure 1
, mutant galectin-3 failed to protect BT549 cells against genistein-induced cytotoxicity, further substantiating the observation that the NWGR motif is critical for galectin-3's ability to prevent cell death.
Galectin-3 inhibits genistein-induced apoptosis and results in cell cycle arrest at G2/M
To determine the mode of genistein-induced cell death, we examined nuclear morphology following genistein treatment. Genistein-treated BT549neo and BT549Galm cells underwent apoptotic changes, including chromosome condensation and fragmentation. In contrast, no significant change was observed in BT549-Galwt cells following genistein treatment (Figure 2A
), suggesting that galectin-3 inhibits genistein-induced apoptosis. To confirm this, we measured caspase activity using the fluorogenic substrate Ac-DEVD-amc, a substrate for caspases 3, 6, 7, 8 and 10. Caspases are a family of cysteine proteases and their activation is regarded as the molecular instigator of apoptosis (30). Caspase activity in BT549neo and BT549-Galwt cells was determined by release of aminomethyl coumarin from the tetrapeptide substrate Ac-DEVD-amc. While genistein significantly induced DEVDase activity in BT549neo, there was no induction in BT549-Galwt cells. These results show that galectin-3 inhibits genistein-induced caspase activity and apoptosis in BT549 cells.

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Fig. 2. Galectin-3 inhibits genistein-induced apoptosis. (A) BT549neo, BT549-Galwt and BT549-Galm cells were treated with 90 µM genistein for 48 h and analyzed for nuclear morphology using bisbenzimide staining. Arrows, apoptotic nuclei. Higher magnifications of the fragmented apoptotic nuclei of BT549neo and BT549-Galm cells are shown in the top, right corner. (B) BT549neo and BT549-Galwt cells were treated with 90 µM genistein for 48 h and DEVDase activity was assayed using Ac-DEVD-amc as a substrate. DEVDase activity was normalized per µg protein. The error bars represent standard deviation of the mean of triplicates.
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We have previously showed that galectin-3 inhibition of apoptosis is associated with its ability to arrest the cell cycle: cisplatin treatment or loss of cell anchorage induces cell cycle arrest at late G1 in galectin-3-overexpressing cells, while it rapidly induces apoptosis in control cells (27,28). Next, we asked if the lack of apoptosis in BT549-Galwt cells following genistein treatment is also associated with galectin-3 involvement in cell cycle regulation. Genistein treatment of BT549neo cells resulted in a rapid decrease in the G0/G1 population (Figure 3A
), and an increase in S phase and sub-G1 (apoptotic) populations, suggesting that genistein triggers both cell cycle entry and apoptosis in these cells. However, genistein treatment reduced the G2/M population of BT549neo cells. This suggests that these cells underwent apoptotic cell death before they reach the G2/M phase. Genistein treatment of BT549-Galwt cells also induced cell cycle entry, as detected by a reduction in G0/G1 population (Figure 3B
). In contrast to BT549neo cells, no increase in the sub-G1 population was detected in genistein-treated BT549-Galwt cells. Instead, with increased exposure time to genistein, BT549-Galwt cells accumulated at G2/M, with reduced G0/G1 and S populations. These studies demonstrate that galectin-3 is a critical determinant for genistein-induced apoptosis and cell cycle arrest at G2/M in BT549 cells.

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Fig. 3. Galectin-3 mediates cell cycle arrest at G2/M arrest. Flow cytometric cell cycle histograms of BT549neo (A) and BT549-Galwt (B) treated with 90 µM genistein for 0, 16, 27 and 40 h. The proportions of cells in each cell cycle phase are presented.
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p21WAF1/CIP1 induction is associated with genistein-induced G2/M arrest, but not with apoptosis
We previously reported that galectin-3 inhibition of anoikis involves cell cycle arrest at late G1 through induction of cyclin D1 (an early G1 cyclin) and cyclin-dependent kinase inhibitors (p21WAF1/CIP1 and p27KIP1) (28). To understand the molecular basis for genistein-mediated cell cycle arrest at G2/M in BT549-Galwt cells, the effect of galectin-3 expression on gene expression of cell cycle regulators was examined. Galectin-3 expression resulted in a basal level increase in cyclin D1, p21WAF1/CIP1 and p27KIP1 as shown in Figure 4A
. Genistein treatment further enhanced expression of cyclin D1 in BT549-Galwt cells (Figure 4B
), which is consistent with increased cell cycle entry following genistein treatment. It should be noted that expression of INK-family inhibitors (p15INK4B, p16INK4A and p19INK4D), known to inhibit cyclin D1 activity, was neither detected nor altered in BT549-Galwt cells (data not shown). While p21WAF1/CIP1 is a universal inhibitor of cyclin-dependent kinases and causes cell cycle arrest at G1/S or at G2/M, p27KIP1 is mostly involved at the G1/S checkpoint, but not G2/M (31). Although galectin-3 increased the basal level of p27KIP1, p27KIP1 expression is drastically down-regulated in BT549-Galwt cells following genistein treatment (Figure 4C
), in agreement with rapid cell cycle progression beyond the G1/S checkpoint. Genistein treatment of BT549-Galwt cells further induced p21WAF1/CIP1 expression as shown in Figure 4B
. Collectively, these results suggest that galectin-3 induction of p21WAF1/CIP1 is in part responsible for genistein-mediated cell cycle arrest at G2/M in BT549 cells. It should be noted that genistein had no effect on p21WAF1/CIP1 expression in BT549neo and BT549-Galm cells, both apoptosis-prone cells. This clearly suggests that p21WAF1/CIP1 expression is not required for genistein-induced apoptosis.

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Fig. 4. Galectin-3 induction of p21WAF1/CIP1 is associated with G2/M arrest. Immunoblot analysis of galectin-3, cyclin D1, p21WAF1/CIP1, p27KIP1 and ß-actin in BT549neo, BT549-Galwt or BT549-Galm cells. Protein samples were prepared from cells without treatment (A) or with 90 µM genistein treatment for the indicated times (B and C).
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Discussion
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Increasing evidence suggests that apoptosis regulation is tightly linked to cell cycle regulation. Although apoptosis can be induced at any point during the cell cycle, apoptosis sensitivity varies greatly at different points in the cell cycle. Genistein has previously been shown to induce cell cycle arrest at G2/M and apoptosis in many cancer cell lines (4,10,16). It has been suggested that p21WAF1/CIP1 is critical for both genistein-induced cell cycle arrest and apoptosis. The present study, however, indicates that genistein-induced p21WAF1/CIP1 may be associated with apoptosis inhibition through cell cycle arrest rather than apoptosis induction. Our study clearly suggests that the level of expression of anti-apoptotic gene products such as galectin-3 is a critical determinant of genistein-induced cell cycle arrest or apoptosis. This is of particular importance in the light of the recently reported potential carcinogenic activity of genistein (14,15). Genistein-induced apoptosis seems to be accompanied by induction of cell cycle entry (Figure 3
). When genistein-induced apoptosis is inhibited by galectin-3, galectin-3 induction of cyclin D1 and rapid cell cycle progression beyond the S phase may enhance accumulation of genetic mutations contributing to carcinogenesis.
Previous studies and the present one suggest that galectin-3 results in cell cycle arrest at different points depending on the apoptotic stimuli. Galectin-3 induces cell cycle arrest at late G1 in response to cisplatin treatment or loss of cell adhesion (anoikis) (27,28), whereas it induces G2/M arrest following genistein treatment. At present it is not known how galectin-3 modulates the expression of cell cycle regulatory genes, including cyclin D1, p21WAF1/CIP1 and p27KIP1. Galectin-3 is expressed in the nucleus and cytoplasm and also in secreted form. Galectin-3 in the nucleus may be involved in the regulation of gene expression. It is equally possible that cytoplasmic galectin-3 or extracellular galectin-3 induces signal transduction leading to modulation of gene expression. Cisplatin treatment or anoikis further enhanced p21WAF1/CIP1 and p27KIP1 expression in galectin-3-expressing cells, resulting in G1/S arrest. In contrast, genistein enhanced p21WAF1/CIP1 expression only, while it abolished galectin-3 induction of p27KIP1, leading to G2/M arrest. Specific interactions between apoptosis initiation signaling and galectin-3 regulation of gene expression remain to be fully investigated.
In summary, the present study provides a mechanistic insight into genistein-induced cell cycle arrest or apoptosis regulated by the anti-apoptotic gene product galectin-3. Taken in conjunction with the finding that galectin-3 is often overexpressed in human cancer (1820,2225), our finding may be critical for understanding the chemopreventive/chemotherapeutic potentials of genistein.
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Notes
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4 Present address: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA 
5 To whom correspondence should be addressed Email: hrckim{at}med.wayne.edu 
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Acknowledgments
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We thank Dr Erik Thompson of the Lombardi Cancer Center for the gift of the BT-549 parental cells, and Ms Mary Ann Krug for preparation of this manuscript. This work was supported in part by grants CA64139 from the NIH/NCI and by DAMD17-99-1-9442 from the US Army (to H.-R.C.K.) and by a grant from Ajou University (to B.M).
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Received May 5, 2000;
revised July 19, 2000;
accepted July 27, 2000.