NUTRITION AND CANCER, 57(1), 48–58 C 2007, Lawrence Erlbaum Associates, Inc. Copyright
Effects of Flavonoids Genistein and Biochanin A on Gene Expression and Their Metabolism in Human Mammary Cells Young Jin Moon, Daniel A. Brazeau, and Marilyn E. Morris
Abstract: Genistein (GEN) and biochanin A (BCA), dietary isoflavones, possess breast cancer–preventive properties. Our objective was to examine the effect of physiologically relevant concentrations of BCA and GEN on gene expression in normal (HMEC), immortalized but nontumorigenic (MCF12A), and tumorigenic (MCF7) mammary cells and to determine whether the differences in gene expression are related to differences in metabolism in the three types of mammary cells. Using cDNA arrays, we compared the gene expression after a 48-h incubation with 1 µM BCA, GEN, or vehicle. Treatment with GEN or BCA produced the greatest number of significant changes in HMEC compared with MCF12A or MCF7 cells. Unlike GEN, effects of BCA on gene expression were mostly beneficial, involving induction of tumor suppressor genes. Different extents of metabolism were observed in the three mammary cell types; however, GEN concentrations were very low following either GEN or BCA administration in all of the three cell types. Because there were only very low concentrations of GEN, compared with BCA concentrations, in HMEC and MCF12A cells treated with BCA and different gene expression changes were found after BCA and GEN treatment, these findings suggest that BCA has distinct effects compared with GEN. The results suggest that BCA may represent a better breast cancer–preventive agent than GEN.
Introduction Breast cancer is the most common cancer in women and the second leading cause of cancer-related death in American women (1). Asian women have a relatively lower incidence of breast cancer. One distinct aspect of Asian diets is the high consumption of soy products. Regular consumption of soy foods by Asian women has been correlated with a lower incidence of breast cancer (2). It has been suggested that the isoflavones provide at least part of the protective effect of soy (3). A predominant isoflavone found in soy is genistein (GEN, 4 ,5,7-trihydroxyisoflavone, Fig. 1). Both in vivo and in vitro studies have shown that GEN is a promising agent for
cancer prevention and/or treatment (4). GEN has pleiotropic molecular mechanisms of action affecting cell cycle regulation, cell growth, angiogenesis, invasion, metastasis, and apoptosis (4). Biochanin A (BCA) is a 4 -O-methyl derivative of GEN (Fig. 1), not present in soy foods, but rather is the major isoflavone constituent in red clover (Trifolium pratense). Popularly sold herbal supplements contain high amounts of BCA (5). The biological effects of BCA have been much less documented compared with those of GEN, but BCA also has cancer-preventive properties (6,7). Even though BCA is converted to GEN in vivo and in vitro, the growth-inhibitory effects of BCA are not identical to those of GEN (8,9). BCA exerts stronger inhibitory effects than GEN in MCF7 cells at high concentrations, which may be due to the greater hydrophobicity of BCA, compared with GEN, increasing its cellular uptake and/or cellular distribution (10). Whether the active constituent in vivo is the parent compound or a metabolite has not been established. The mechanism by which these compounds inhibit proliferation and induce apoptosis in cancer cells has not been fully elucidated. The chemical structures of isoflavones are similar to 17β-estradiol (Fig. 1), and it is known that isoflavones can weakly bind to the estrogen receptor (ER) (11). BCA and GEN show a biphasic effect on the growth of ER-positive human breast cancer cells, inhibiting at supraphysiological concentrations (>20 µM) through ER-independent mechanisms while stimulating at lower concentrations (6). Allred et al. have suggested that GEN could increase mRNA levels of the estrogen-responsive gene pS2 in a dose-dependent manner (12). Although studies have been performed using high concentrations (>20 µM) of isoflavones, the maximum plasma concentration of any flavonoid rarely exceeds 1 µM following dietary intake (13). To better understand the precise molecular mechanism(s) by which isoflavones exert their effects on breast cancer cells, we utilized cDNA gene arrays to assess the gene expression profiles of breast cancer cells treated with a physiologically relevant level (1 µM) of BCA and GEN, three kinds of mammary cells. MCF7 cells (a human breast cancer cell line), MCF12A cells (a
All authors are affiliated with the Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Amherst, New York
Figure 1. Chemical structure of 17β-estradiol, biochanin A, and genistein.
nontumorigenic human mammary epithelial cell line), and human mammary epithelial cells (finite lifespan 184 HMEC), were used in this investigation. Although considerable attention has been focused on examining the inhibition of proliferation of breast cancer cell lines such as MCF7, much less is known about isoflavone effects on normal finite lifespan mammary epithelial cells (HMEC) or breast epithelial cells at earlier stages in the oncogenic process (MCF12A). The effects of isoflavones on these cells may be more relevant in understanding the cancer-preventive effect. Materials and Methods Materials BCA, GEN, transferrin, isoproterenol, diethyl ether (high-performance liquid chromatography grade), and β-glucuronidase type H-5 (aryl-sulfate sulfohydrolase from Helix pomatia; reported sulfatase activity of 15–40 units/mg and glucuronidase activity of 400–600 units/mg) were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s modified Eagle’s medium and Ham’s F12 medium, RPMI 1640 medium, fetal bovine serum (FBS), horse serum, and phosphate-buffered saline (PBS) were from Invitrogen (Grand Island, NY). Mammary Epithelia Basal MCDB 170 Medium (sodium bicarbonate–free) and bovine pituitary extract were from Clonetics (San Diego, CA). MCF7 cells were obtained from the National Cancer Institute (Frederick, MD), MCF12A cells were obtained from the American Type Culture Collection (Manassas, VA), and HME cells were kindly provided by Dr. Martha Stampfer (Lawrence Berkeley National Laboratory, Berkeley, CA). MCF7 cells are a human breast cancer cell line. MCF12A cells are a nontumorigenic human mammary epithelial cell line established from tissue taken at reduction mammoplasty from a 60-yr-old nulliparous patient with fibrocystic breast disease that contained focal areas of intraductal hyperplasia. HME cells are human mammary epithelial cells. We used normal finite lifespan 184 HME cells that were obtained from reduction mammoplasty tissue of a 21-yr-old woman. They senesce around p22, when cultured in serum-free MCDB 170 medium (14). Antibodies were purchased from Cell Signaling (Beverly, MA).
Cell Culture MCF7 cells were grown in 75-cm2 cell culture flasks in RPMI 1640 culture media supplemented with 10% FBS, Vol. 57, No. 1
100 units/ml penicillin, and 100 µg/ml of streptomycin in a 37◦ C incubator in a humidified atmosphere of 5% CO2 /95% air. MCF12A cell lines were maintained in 75-cm2 cell culture flasks in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium, 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 0.01 mg/ml insulin, 500 ng/ml hydrocortisone, and 5% horse serum in a 37◦ C incubator in a humidified atmosphere of 5% CO2 /95% air. HME cells were cultured in 100-mm dishes in Mammary Epithelia Basal Medium, 5 ng/ml epidermal growth factor, 500 ng/ml hydrocortisone, 5 µg/ml insulin, 70 µg/ml bovine pituitary extract, 5 µg/ml transferrin, and 10−5 M isoproterenol in a 37◦ C incubator in a humidified atmosphere of 1% CO2 /99% air. MCF7 cells with passage number of 16–24, MCF12A cells with passage number of 55–56, and HME cells with passage number of 9–10 were used for experiments. Cells were treated with 1 µM GEN, 1 µM BCA, or dimethyl sulfoxide (DMSO, vehicle control) for 48 h. The rationale for choosing this time point was to capture gene expression profiles of genes involved during the onset of growth inhibition and apoptotic processes. The concentration level of isoflavones used is one that is achievable in plasma after the consumption of food.
Total RNA Isolation Total RNA from each sample was isolated using the SV Total RNA Isolation System (Promega, Madison, WI) per the manufacturer’s instruction. RNA was quantitated spectrophotometrically at 260 nm.
Gene Array Two specific human GEArray Kits (SuperArray, Bethesda, MD), signal transduction pathway array and cancer/tumor suppressor array, were utilized. Each array consists of 23 genes spotted in duplicate as well as control spots (PUC18 as negative control; β-actin and glyceraldehyde-3-phosphate dehydrogenase, G3PDH). The gene arrays were used according to the manufacturer’s instructions. In brief, using the reagents provided, genespecific cDNAs were prepared and labeled from total RNA by reverse transcription with mouse mammary tumor virus (MMLV) reverse transcriptase (Invitrogen, Grand Island, NY) and chemiluminescence-labeled biotin deoxyuridine 5 triphosphate (dUTP) (Invitrogen). Relative expression levels of each gene were analyzed using a Kodak Image Station 440CF (Eastman Kodak Company, Rochester, NY). For normalization, we chose total intensity excluding βactin and G3PDH, which has the lowest gene-stability measure (Mj ) (15) compared with the provided housekeeping gene spots. Each experiment was repeated three to four times. 49
Table 1. Primer Sequences and Reaction Conditions for Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction Gene name (GenBank accession no.) PTEN (U96180) egr-1 (X52541) β-Actin (X00351)
Forward Primer (5 -3 )
Reverse Primer (5 -3 )
Annealing Temperature (◦ C)
Predicted Size (bp)
TCT GAG TCG CCT GTC ACC ATT AAC GCA AGA GGC ATA CCA AGA T CTG GCC GGG ACC TGA CT
CCG TGT TGG AGG CAG TAG AAG CCG AAG AGG CCA CAA CAC TT TCC TTA ATG TCA CGC ACG ATT T
57 57 57
78 75 100
Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction Real-time quantitative reverse transcriptase-polymerase chain reaction (RTQ RT-PCR) was performed on β-actin (for normalization), PTEN, and egr-1 using the Stratagene Mx4000TM Multiplex Quantitative PCR System (Stratagene, La Jolla, CA). The same total RNA prepared for the gene arrays was also used for RTQ RT-PCR. Total RNA (600 ng) from each sample was reverse transcribed into cDNA using a Superscript first strand cDNA synthesis kit (Invitrogen) according to the manufacturer protocol. PCR reactions for phosphatase and tensin homolog (PTEN), egr-1, and β-actin were carried out by mixing 5 µl of cDNA, 5 µl of 10× PCR buffer, 2 µl of deoxynucleoside triphosphate mix (5 mM each dATP, dCTP, dGTP, and dTTP), 1 µl each of 10 µM primer, 0.5 µl reference dye rhodamine-X (1/500 dilution, Molecular Probes, Eugene, OR), 0.5 µl SYBR green I (1/750 dilution, Molecular Probes), 2 U Taq polymerase (Eppendorf, Westbury, NY), and 34.75 µl H2 O and amplified for 35 cycles. Primers (Table 1) were designed using the computer pro R gram Primer Express (Perkin-Elmer Applied Biosystems, Foster City, CA). The PCR products were resolved by electrophoresis through a 2% agarose gel to confirm target size and presence of single PCR product. R The PCR product of each gene was cloned into a pCR R 2.1 TOPO vector (Invitrogen) and transformed into One Shot chemically competent Escherichia coli cells (Invitrogen). Cloned PCR products were confirmed by sequencing and used to construct standard curves for absolute quantification of copy number. The standard curves were run in triplicate concurrently on the same plate with samples, also in triplicate. The reported copy number was estimated from the linear regression of the standard curve on the same plate.
Western Analysis of PTEN and Phospho-Akt (Ser473) Cells were washed with PBS and harvested using a rubber policeman. Total cell lysates were prepared by adding the lysis buffer (20 mM Tris pH 7.5, 120 mM NaCl, 100 mM NaF, 1% Nonidet P-40, 200 µM sodium orthovanadate, 50 mM β-glycerolphosphate, 10 mM sodium pyrophosphate, 4 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) to the harvested cells. The cells were kept on ice for 30 min. The soluble 50
extracts were obtained by centrifuging the cell lysates at 13,000 g for 20 min. The protein concentrations of the soluble extracts were obtained by bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL). Proteins (50 µg) were electrophoresed on 7.5% sodium dodecyl sulfate–polyacrylamide gels and electroblotted onto nitrocellulose membranes (Invitrogen, Grand Island, NY). Membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.2% (vol/vol) Tween 20 and 5% (wt/vol) fat-free dry milk (Bio-Rad, Hercules, CA) and then incubated first with primary antibody overnight at 4◦ C and then with secondary antibody at room temperature for 1 h. Anti-mouse IgG horseradish peroxidase (Amersham Biosciences, Piscataway, NJ) was used as secondary antibody. As a loading control, some of the membranes were also incubated with anti-β-actin antibody (Alltech Associates, Deerfield, IL) and anti-mouse IgG to detect β-actin. After incubation with the antibodies, membranes were washed and detected with enhanced chemiluminescence detection reagent (Amersham Biosciences). The Kodak Image Station 440CF (Kodak) was use to analyze the Western blot results.
Statistical Analysis One-way analysis of variance followed by Newman-Keuls test was used for evaluating differentially expressed genes among the three types of cells using gene arrays. For comparisons of two groups, Student’s unequal variance t-test with P < 0.05 set as the significance level was used for statistical analysis for both arrays and RTQ RT-PCR.
Metabolism Studies BCA, GEN, or DMSO (vehicle control) was added to the cells to a final concentration of 1 µM to assess metabolism of the compounds. All experiments were carried out in triplicate. After 48-h incubations, media were aspirated, and the cells were washed with 10 ml of cold 1× PBS three times. Cells were harvested by scraping and lysed by Branson R SONIFIER ultrasonic cell disruptor (Branson Ultrasonics Corporation, Danbury, CT) in 400 µM of double-distilled water. “Total” isoflavones (free aglycones plus aglycones released from glucuronide and/or sulfate conjugates) were measured by using glucuronidase/sulfatase type H-5 (from Nutrition and Cancer 2007
Table 2. Differentially Expressed Genes Among the Three Cell Typesa Gene Name Description APC CBP DPC4
NF2 p300 P53 TGFβR2
egr-1 gadd45
Hsp27 NFκB
pig8
HMECb
Adenomatosis polyposis coli 1.24 ± 0.777 Human CREB-binding 1.93 ± 0.800 protein 1.33 ± 0.312 Mothers against decapentaplegic homolog 4 (Drosophila) Neurofibromin 2 2.25 ± 1.26 E1A-binding protein p300 1.72 ± 0.422 Tumor protein p53 1.77 ± 0.321 1.75 ± 0.521 Transforming growth factor, beta receptor II (70–80 kDa) Early growth response 1 1.27 ± 0.250 Growth arrest and 1.43 ± 0.218 DNA-damage-inducible transcript 1 Heat shock 27-kDa protein 1 5.31 ± 0.332 Nuclear factor of kappa light 0.806 ± 0.0409 polypeptide gene enhancer in B-cells 1 Etoposide-induced mRNA 0.805 ± 0.156
MCF12Ab
MCF7b
MCF12A/HMECc MCF7/HMECc MCF7/MCF12Ac
8.69 ± 1.73 1.27 ± 0.449
8.36∗ 0.211∗
7.00* 0.658
0.837 3.12
0
0.112 ± 0.058
—∗∗d
0.0844∗∗
0.0844/d
0.300 ± 0.137 0.402 ± 0.252 0.103 ± 0.106 0.407 ± 0.360
0.880 ± 0.119 0.532 ± 0.0674 0.754 ± 0.306 0.0461 ± 0.0535
0.133∗ 0.234∗ 0.0583∗∗ 0.233∗∗∗
0.390 0.309∗ 0.427∗∗∗ 0.0264∗∗∗
2.93 1.32 7.32∗ 0.113
1.08 ± 0.317 0.613 ± 0.058
0.480 ± 0.209 0.510 ± 0.284
0.847 0.428∗∗∗
0.378∗ 0.356∗∗∗
0.446∗ 0.831
3.90 ± 1.06 5.96 ± 0.140 0.680 ± 0.0697 0.391 ± 0.155
0.733∗ 0.844
1.12 0.485∗∗∗
1.53∗ 0.574∗
0.580 ± 0.231
0.720
0.328∗
0.456
10.4 ± 5.86 0.407 ± 0.222
0.264 ± 0.0943
a : n= 3 or 4; *P < 0.05; ∗∗ P < 0.001; ∗∗∗ P < 0.01. Significant differences are shown in bold. Three types of mammary cells were treated with the vehicle (dimethyl sulfoxide) for 48 h. Arrays were normalized to total intensity (without β-actin and glyceraldehyde-3-phosphate dehydrogenase). b : Results are presented as normalized intensities (mean ± SD). c : Results are presented as ratios of the normalized average values for two cell types. d : No expression was measured in control MCF12A.
H. pomatia, Sigma Chemical, St. Louis, MO) as the deconjugation enzymes. One hundred µM of 0.2% sodium chloride containing sulfatase/glucuronidase (100/1,000 units) and 200 µM of 0.2 M sodium acetate (pH 5.0 at 37◦ C) were added to the 100 µM of cell lysate and incubated at 37◦ C for 3 h. Samples were extracted with 1 ml diethyl ether, and the organic layers were evaporated to dryness with nitrogen. Dried extracts were reconstituted in 70 µM of 10 mM ammonium acetate/1% isopropyl alcohol/0.1% formic acid:acetonitrile at a 1:1 ratio and injected into the liquid chromatography/mass spectrometry/mass spectrometry system. Free BCA and GEN concentrations were measured in the same fashion as described for the total BCA and GEN except that 100 µM of 0.2% sodium chloride without enzymes was added to the samples. Isoflavone sulfate and/or glucuronide conjugate levels were determined by subtracting the free aglycone level from the level determined after glucuronidase/sulfatase digestion. The BCA protein assay (Pierce Chemical) was used to determine total cellular protein, and this was used to normalize the cellular drug concentrations measured by LC/MS/MS. Estimation of protein was performed after cell lysis by sonication. One milliliter of working reagent was added to 50 µl of cell lysate followed by measurement of the optical density at 562 nm after a 30-min incubation. LC/MS/MS System The analysis was carried out on a PE Sciex API3000 triple-quadropole mass spectrometer with a turbo ion spray Vol. 57, No. 1
source linked to a Shimadzu LC10 LC system equipped with an Advantage Armor column (C18, 30 mm × 4.6 mm inner diameter, 5 µm, Analytical Sales and Service, Pompton Plains, NJ). The source temperature was set at a constant 450◦ C. The mobile phase consisted of 1) acetonitrile and 2) 10 mM ammonium acetate +1% isopropyl alcohol. The sample was eluted using a gradient from 0 to 95% acetonitrile at 0.75 ml/min. The ions measured for BCA were 283/239 and for GEN 269/133. BCA eluted at 2.03 min and GEN eluted at 1.79 min. The lower limit of quantification was 1 ng/ml for both BCA and GEN. Standards and quality controls were included with samples for every run so that intra- and interday variability was adjusted with the standards. The interday variability for the entire standard curve was ∼17% for BCA and ∼11% for GEN.
Results Gene Expression Differences Among Cell Types Twenty-two genes showed significantly different expression patterns among the three cell types (Table 2). The least number of differences in significantly altered genes was observed comparing MCF7 and MCF12A cells. Five tumor suppressor genes (DPC4, p300, p53, TGFβR2, and gadd45) showed decreased expression in MCF12A and MCF7 cells compared with HMEC. The expression of 30 genes (ATF-2, bax, BRCA1, BRCA2, CD5, c-fos, c-myc, CYP19, IRF-1, 51
Table 3. Significantly Changed Gene Expression Following Treatment with 1 µM of BCAa HMEC Cell Type Gene Name p18 ATF-2 CD5 IκBα
MSH2 NF2 p27Kip1 WT1
MCF12A
Description
Controlb
BCAb
BCA/ Controlc
Cyclin-dependent kinase inhibitor 2C (inhibits CDK4) Activating transcription factor 2 T-cell surface glycoprotein CD5 Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha mutS (E. coli) homolog 2 (colon cancer, nonpolyposis type 1) Neurofibromin 2 Cyclin-dependent kinase inhibitor 1B Wilms’ tumor 1
0.237 ± 0.211
0.776 ± 0.102
3.27∗
0.895 ± 0.234 0.896 ± 0.319 0.644 ± 0.212
1.37 ± 0.153 1.18 ± 0.220 0.690 ± 0.286
1.53∗ 1.31 1.07
0.587 ± 0.626
0.852 ± 0.188
1.45
2.25 ± 1.26 0.617 ± 0.435
0.951 ± 0.336 0.574 ± 0.372
0.143 ± 0.129
0.981 ± 0.243
Controlb
BCAb
0.100 ± 0.104
BCA/ Controlc
0.160 ± 0.171
1.60
1.00 ± 0.513 1.85 ± 0.810 1.12 ± 0.462
1.18 3.40∗ 2.98∗
0.573 ± 0.304
1.24 ± 0.244
2.16∗
0.422 0.931
0.300 ± 0.137 0.409 ± 0.0149
0.855 ± 0.274 0.0362 ± 0.0562
6.85∗
0.274 ± 0.0901
0.170 ± 0.255
0.847 ± 0.0280 0.543543 ± 0.149 0.377 ± 0.260
2.85∗ 0.0886∗∗ 0.622
a : BCA, biochanin A. n = 3 or 4; *P < 0.05; **P < 0.01. Significant differences are shown in bold. b : Results are expressed as normalized intensity (mean ± SD) compared with vehicle controls and are the average of three or four independent macroarray experiments. c : Results are expressed as fold change compared with vehicle controls and are the average of three or four independent macroarray experiments.
MSH2, Fas, p18, p19, p21, p27Kip1, hsp1, hsp90, IkBa, IL-2, iNOS, p57kip2, mdm2, PTEN, Rb, TGFbR1, TSC-1, TSC-2, VHL, WT1, and pig7) was not significantly different in all three cell types. Effect of BCA and GEN on Gene Expression in Different Cell Types BCA (1 µM) treatment significantly altered the expression of three genes in HME cells and five genes in MCF12A cells. In contrast, no genes were affected by BCA treatment in MCF7 cells (Table 3). BCA increased the expression of tumor suppressor genes MSH2 and NF2 in MCF12A cells and WT1 in HME cells. MSH2 is known to be involved in DNA mismatch repair. The function of the WT1 protein in breast cancer is not known. Cyclin E is a target of WT1 transcriptional repression (16). WT1 negatively regulates the expression of insulin-like growth factor-I receptor gene, which plays a critical role in transformation (17). The expression of p18, which suppresses cell growth, was up-regulated by BCA in normal cells. BCA also increased expression of IκBα, which regulates tumorigenesis and apoptosis signals as an inhibitor of NFκB (18). Treatment with 1 µM GEN resulted in more alterations in gene expression than that observed with BCA (Table 4). In normal HMEC cells, GEN exposure resulted in the down-regulation of hsf1, hsp27, and egr-1 and up-regulation of gadd45 and IRF-1. HSF-1 controls hsp90 expression by binding heat shock element promoter (19). In murine uterine tissue, HSF-1 is activated by estrogen treatment. Hsp27 is involved in inhibition of apoptosis in NFκB and p53 signaling pathways. IκBα is degraded by HSP27 through the 26s proteasome (20). egr-1 plays a role in the mitogenic proliferation signal and enhances tumor growth (21). gadd45 is a cell cycle arrest gene, and IRF-1 is tumor suppressor gene. The 52
expression of tumor suppressor genes (ATF-2, DPC4, p53, pig8, TGFβR, and VHL), cell cycle arrest genes (p19 and p21), and the pro-apoptotic gene (bax) was down-regulated in HME cells, whereas the expression of hsp90 and mdm2 was up-regulated in HME cells. These alterations of these 11 genes by GEN would be generally undesirable for a cancerprotective agent. BCA and GEN had similar effects as well as contrasting effects on gene expression in each of the cell lines. Both BCA and GEN increased expression of CD5 and NF-2 in MCF12A. The function of CD5 in breast cancer is not known, but multiple somatic mutations of CD5 were found in Bcell leukemia and lymphoma (22). Mutations affecting both isoforms of the NF2 transcript were detected in multiple tumor types including melanoma and breast carcinoma (23). BCA and GEN had opposite effects on ATF-2 expression. Although expression of ATF-2 in HME cells was increased by BCA, it was decreased by GEN. Breast cancer frequently develops in heterozygous mutant mice for the ATF-2 gene. Therefore, the ATF-2 gene is considered a candidate tumor suppressor gene (24). HMEC cells exhibited the greatest alteration in gene expression following GEN treatment compared with the two other cell lines. Undesirable changes with regard to cancer prevention were more common in HMEC than in MCF12A and MCF7 cell lines. Following exposure to GEN, the expression of tumor suppressor genes (ATF-2, DPC4, p53, pig8, TGFβR, and VHL), cell cycle arrest genes (p19 and p21), and the pro-apoptotic gene (bax) was all down-regulated in HME cells. RTQ RT-PCR of PTEN and egr-1 mRNA Expression Among the significantly changed gene expression, we selected PTEN and egr-1, two of the newly identified Nutrition and Cancer 2007
53
Description
0.946 ± 0.652 0.278 ± 0.280 0.402 ± 0.252 0.580 ± 0.231 0.519 ± 0.257 0.317 ± 0.115 0.407 ± 0.360 2.10 ± 1.26
0.213∗∗∗ 0.161∗ 0.743 0.455∗ 11.8∗∗∗ 0.734/d 0.249∗ 0.180∗
0.599 ± 0.022 0.128 ± 0.074 0.284 ± 0.488 1.28 ± 1.34 0.366 ± 1.88 11.7 ± 0.840 0 0.435 ± 0.312 0.210 ± 0.170
1.77 ± 0.321 1.72 ± 0.422 0.805 ± 0.156 0.994 ± 0.972 0.734 ± 0.915 1.75 ± 0.521 1.17 ± 0.428
0.219 ± 0.173
0.913 ± 0.497 3.90 ± 1.06 6.42 ± 2.33 0 0.489 ± 0.116
0.0805∗ 0.251∗∗∗ 3.48∗∗∗ 4.95∗ 8.85∗∗
0.549 ± 0.195 0.0902 ± 0.105 0.164∗
0.071 ± 0.052 1.34 ± 0.658 10.4 ± 1.50 0.604 ± 0.181 4.03 ± 0.992
0.888 ± 0.172 5.31 ± 0.332 2.99 ± 0.746 0.122 ± 0.212 0.455 ± 0.117
1.08 ± 0.317 0.613 ± 0.058
0.0912∗ 2.02∗∗
0.300 ± 0.137 0.100 ± 0.104
0.116 ± 0.035 2.90 ± 0.537
1.27 ± 0.250 1.43 ± 0.218
10.4 ± 5.86 0.847 ± 0.028 0.505 ± 0.141 3.19 ± 2.73 0.543 ± 0.149 0
Controlb
0.248 0.146∗ 0.452∗ 0.297 0.791 0.0979∗∗
GEN/Controlc
0.122 2.57
0.308 ± 0.515 0.131 ± 0.168 0.192 ± 0.071 0.146 ± 0.253 0.709 ± 0.653 0.130 ± 0.225
1.24 ± 0.777 0.895 ± 0.234 0.425 ± 0.109 0.493 ± 0.472 0.896 ± 0.319 1.33 ± 0.312
2.25 ± 1.26 0.274 ± 0.450 0.237 ± 0.211 0.610 ± 0.723
GENb
Controlb
0.880 ± 0.119 0.0598 ± 0.008
5.72∗ 12.0∗
1.62 ± 0.278
0.201 ± 0.145 0.565 ± 0.612 0.610 ± 0.226 1.89 ± 0.478 1.03 ± 0.254 2.79 ± 1.57
1.91 ± 0.606
0.770
0.724 1.40 1.05 3.64∗ 3.25∗ 6.84
2.02
1.64 ± 0.805
0.585 ± 0.442 0.532 ± 0.067 0.264 ± 0.094 0.249 ± 0.129 0.139 ± 0.063 0.046 ± 0.054
0.632 ± 0.199
0.146 ± 0.0934
1.61 ± 0.252 5.96 ± 0.140 6.90 ± 1.30 0.0176 ± 0.0161 0.279 ± 0.176
0.480 ± 0.209 0.510 ± 0.284
8.69 ± 1.73 0.782 ± 0.487 0.559 ± 0.320 4.44 ± 0.668 0.844 ± 0.210 0.112 ± 0.058
Controlb
0.936 1.16 0.716 0.427/d 1.63
1.18 1.97
0.138 1.14 0.600 0.150 2.17∗ 0.838d
GEN/Controlc
0.878 ± 0.167 4.00∗∗
1.72 ± 0.601 1.19 ± 0.292
0.855 ± 0.558 4.51 ± 0.719 4.59 ± 2.07 0.427 ± 0.181 0.795 ± 0.590
1.27 ± 0.957 1.21 ± 0.545
1.43 ± 0.290 0.969 ± 0.397 0.303 ± 0.182 0.479 ± 0.730 1.18 ± 0.293 0.838 ± 0.458
GENb
MCF12A
a : GEN, genistein. n = 3 or 4; ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. Significant differences are shown in bold. b : Results are expressed as normalized intensity (mean ± SD) compared with vehicle controls and are the average of three or four independent macroarray experiments. c : Results are expressed as fold change compared with vehicle controls and are the average of three or four independent macroarray experiments. d : No expression was measured.
Adenomatosis polyposis coli Activating transcription factor 2 BCL2-associated X protein Breast cancer 1, early onset T-cell surface glycoprotein CD5 Mothers against decapentaplegic homolog 4 (Drosophila) egr-1 Early growth response 1 gadd45 Growth arrest and DNA-damage-inducible transcript 1 hsf1 Heat shock transcription factor 1 hsp27 Heat shock 27-kDa protein hsp90 Heat shock 90-kDa protein IRF-1 Interferon regulatory factor 1 mdm2 Mouse double minute 2, human homolog of; p53-binding protein NF2 Neurofibromin 2 p18 (cdk4 inhibitor) Cyclin-dependent kinase inhibitor 2C p19INK4d Cyclin-dependent kinase inhibitor 2D p21Waf1 (p21Cip1) Cyclin-dependent kinase inhibitor 1A (p21, Cip1) p53 Tumor protein p53 p300 E1A-binding protein p300 pig8 Etoposide-induced mRNA PTEN Phosphatase and tensin homolog 1 Rb Retinoblastoma 1 TGFβR2 Transforming growth factor, beta receptor II VHL Von Hippel-Lindau syndrome
APC ATF-2 (creb-2) bax BRCA1 CD5 DPC4
Gene Name
HMEC
Table 4. Significantly Changed Gene Expression Following Treatment with 1 µM of GENa
0.313∗ 0.873 0.951 0.318∗ 1.40 5.69
GEN/Controlc
0.643 1.15 0.628 22.6 1.26
2.39 ± 1.19
0.661 ± 0.356 0.720 ± 0.026 1.028 ± 0.160 0.807 ± 0.106 0.613 ± 0.213 0.451 ± 0.357
1.45
1.13 1.35∗ 3.89∗∗ 3.24∗∗ 4.41 9.78
1.356 ± 0.183 2.14∗∗
0.374 ± 0.361 2.57
1.19 ± 0.537 1.35 0.227 ± 0.200 3.79
1.04 ± 1.00 6.86 ± 0.977 4.33 ± 2.21 0.398 ± 0.400 0.352 ± 0.033
0.444 ± 0.305 0.926 0.371 ± 0.335 0.728
2.72 ± 2.55 0.682 ± 0.524 0.532 ± 0.175 1.41 ± 1.38 1.18 ± 0.423 0.637 ± 0.428
GENb
MCF7
RTQ RT-PCR, and 3) some of the samples used in RTQ RTPCR were different from ones used in the original arrays (25). Western Analysis of PTEN and Phospho-Akt (Ser473) Figure 2. Western blot analysis of PTEN and phospho-Akt expression in MCF7 cell vehicle (CON) or 1-µM genistein treatment (GEN) for 2 days. Cellular PTEN and phospho-Akt levels were examined by Western blot analysis, as described in Materials and Methods. β-Actin was used to confirm equal protein loading. Two separate experiments were conducted, and similar results were obtained.
GEN-regulate genes in HME cells, for confirmation using real-time RT-PCR. Standard curves for PTEN and egr-1 were y = −2.968 × log(x) + 0.65 (r2 = 0.933) and y = −3.337 × log(x) + 0.32 (r2 = 0.985), respectively, with the x-axis representing the dilution factor. Standards were diluted from 0.280 mg/ml of PTEN dsDNA and 0.240 mg/ml of egr-1. The y-axis represents threshold cycle number. RTQ RT-PCR confirmed the gene array results demonstrating significant increases in expression of PTEN (1.66-fold; P = 0.002) and decreases in expression of egr-1 (0.683-fold; P = 0.046). The changes were qualitatively but not quantitatively similar between gene array and RTQ RT-PCR. Reasons for the quantitative differences may include 1) the large variability of gene array data caused by small sample numbers, 2) the normalization methods used were different for arrays and
Treatment with 1 µM GEN for 2 days increased PTEN protein expression (∼2 fold) and decreased phospho-Akt protein expression in MCF7 cells, as shown in Fig. 2 (∼0.6 fold). The intensity of the bands was normalized to β-actin. Two separate experiments were conducted, and similar results were obtained. Metabolism Study Low levels of GEN and moderate levels of conjugated GEN were formed in all types of cells treated with BCA (Fig. 3). Interestingly, in all three cell types treated with GEN, more conjugated BCA than conjugated GEN was observed, and no free BCA aglycone was detected. MCF7 cells extensively metabolized both isoflavones. In contrast, less metabolism was observed in MCF12A and HMEC than MCF7 cells. GEN was more susceptible to conjugation than BCA because of its free hydroxyl groups, resulting in very low GEN levels and high conjugated GEN concentrations. BCA was predominantly metabolized directly to conjugated metabolites with some undergoing demethylation to GEN and further metabolism to GEN conjugates.
Figure 3. Concentrations (ng/ml/mg) of isoflavones in three mammary cell types. Metabolism of biochanin A (BCA) and genistein (GEN) was determined by liquid chromatography/mass spectrometry (MS)/MS. BCA or GEN was added to the cells to a final concentration of 1 µM; 48-h incubations. Parent compounds are shown in bold. Results are presented as average concentrations of isoflavone normalized to the amount of cell lysate protein ± SD; n = 3.
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Discussion The purpose of our study was to examine the effect of BCA and GEN on gene expression in three different mammary cells, HMEC, MCF12A, and MCF7, at a physiologically relevant isoflavone concentration (1 µM). We also determined the metabolism of GEN and BCA in these three mammary cell lines. In addition, we compared the basal expression of various genes in the different cell types. We chose these cell lines to evaluate isoflavone effects on cancer prevention, initiation, and progression. HME cells were chosen to investigate cancer-preventive properties of isoflavones on normal finite lifespan cells; immortal transformation, as seen with MCF12A cells, is considered to be an important early transition in the malignant progression of human breast cancer cells, as studied with MCF7 cells. If the gene alteration could cause inhibition, delay, or reversal of carcinogenesis on preinitiated or initiated tumor cells by apoptosis or cell cycle inhibition, or by other actions, this alteration was referred to as “beneficial.”
Cell Type Differences As tumors progress to more advanced stages, they acquire an increasing number of genetic alterations, and this is well demonstrated in our study. Nine genes in tumorigenic MCF7 cell lines were differentially expressed compared with normal HME cells, whereas only three genes were differentially expressed in MCF7 cells compared with MCF12A cells (Table 2). Underexpression of egr-1 (26) is consistent with the results of other studies. egr-1 is known to have a significant role in carcinogenesis and in cancer progression, especially metastasis (27); inhibition of egr-1 resulted in the inhibition of tumor growth in MCF7 xenograft mice (28). All five genes (DPC4, p300, p53, TGFβR2, and gadd45) that were decreased in MCF12A compared with HME cells were also underexpressed in MCF7 cells compared with HME cells. They are all related to tumor suppression and cell cycle arrest. The tumor suppressor genes CBP and NF2 were underexpressed in MCF12A cells compared with HME cells. The alterations of these genes may play a role in the transition of nontumorigenic cells into malignant cells.
Isoflavone Treatment Effects In the present study, the alterations of gene expression were different following treatment with the two isoflavones as well as among the three different types of cells. Unlike GEN, effects of BCA on gene expression were mostly beneficial with regard to cancer prevention, including an increase in a number of tumor suppressor genes (p18, MSH2, NF2, and WT1) in HMEC and MCF12A cells. It is likely that the parent drug exerts these effects because we could detect relatively high concentrations of BCA in HMEC and MCF12A cells. These results suggest that BCA may be a better cancerVol. 57, No. 1
preventive agent than GEN at concentrations that can be obtained following dietary intake. It also suggests that BCA has distinct effects compared with GEN because there were very low concentrations of GEN in HMEC and MCF12A cells. The changes in gene expression were different among the different types of cells. Only five genes (PTEN, p21, p19, pig8, and Rb) that were significantly changed by GEN were the same in the three types of cells evaluated. None of the genes significantly altered by BCA showed consistent patterns of expression among the three different cell types. One of the most important findings of our study may be the up-regulation of PTEN by GEN, which was confirmed by RTQ RT-PCR and Western analysis. A recent study also reported an increase in PTEN expression in rat mammary epithelial cells and MCF7 cells (29). PTEN was significantly up-regulated in all three types of cells by GEN, which represents a beneficial effect with respect to cancer prevention. The main role of PTEN is to negatively regulate Akt activation by preventing its phosphorylation. Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 and by phosphorylation within the carboxyterminus at Ser473 (30). Akt promotes cell survival by inhibiting apoptosis through its ability to phosphorylate and inactivate several targets including Bad (31), Forkhead transcription factors (32), and caspase-9 (33). Our results showed that protein expression of phospho-Akt was reduced by 1 µM of GEN in MCF7 cells, which is consistent with another report (34). PTEN could also increase p27 and decrease protein levels of cyclin D1 (35), which represent opposite actions to those mediated through the estrogen–ER complex (36). Several groups have suggested that one of the mechanisms of the cancer-preventive effects of GEN may be the prevention of Akt activation (37). However, the present work provides evidence for the possible involvement of PTEN (which is directly upstream in the Akt pathway) in the mechanism of action. Moreover, in an additional study, 10 µM of GEN produced a 28.0-fold increase of PTEN compared with a 1.66-fold increase by 1 µM of GEN (Moon YJ and Morris ME, unpublished data). Another important altered gene identified in the present investigation was egr-1; egr-1 expression was decreased by GEN treatment in HME cells. egr-1 mRNA and protein are strongly induced by estrogen in an ER-dependent manner, and both ERα and ERβ have been shown to induce the egr-1 promoter in MCF7 cells and other cancer cell lines (38,39). Therefore, the decreased expression of egr-1 may reflect the antiestrogenic effects of GEN. It is interesting that GEN treatment altered the expression of p19, p21, and pig8 in opposite directions in HMEC when compared with both MCF12A and MCF7 cells. All three genes were down-regulated in HME cells, but p19 was up-regulated in MCF12A cells and p21 and pig8 were upregulated in MCF7 cells. p19 and p21 are involved in cell cycle arrest, and pig8 is involved in apoptosis induction. The reason for these differences is unknown but may involve differences in the basal expression of these genes or upstream 55
genes in these cell lines or differences in GEN metabolism in the three cell lines, among others. Overall, BCA changed the expression of eight genes. Alterations in seven of these genes could be considered beneficial effects for cancer prevention. Only the reduction of p27kip1, a cyclin-dependent kinase inhibitor that results in growth stimulation in MCF12A cells, would be considered a negative effect with regard to cancer prevention. p27Kip1 is down-regulated by estrogens in breast cancer cells through Skp2 and through nuclear export mediated by the extracellular signal-regulated protein kinase (ERK) pathway (40). However, MCF12A has very low expression of ERα and ERβ. Thus, this effect is unlikely to be due to the estrogenic properties of BCA.
cer cells convert isoflavones to phase I and phase II metabolites (9,10,48). In addition, GEN and BCA can be chlorinated and nitrated by inflammatory oxidants, and individual chlorinated isomers show increased binding to ERβ (49). These modifications may influence their binding to ERα or ERβ or to other protein targets. In MCF12A cells, which express little ERα and ERβ (44,50), BCA and GEN still altered gene expression, and most of the effects represented beneficial changes with regard to cancer prevention. Thus, we speculate that at least some of the actions of GEN and BCA may be mediated through mechanisms other than the ER (51), and ER-mediated effects may represent mainly undesirable effects, as observed in HMEC and MCF7 cell lines. Isoflavone Metabolism and Its Relationship with Gene Expression Changes
Relationship with Estrogen Receptor–Mediated Effects Due to the structural similarity of isoflavones to biological estrogens, BCA and GEN can weakly bind to ERs; such binding suggests that they might either induce cell proliferation (estrogenic agonist) or prevent hormone-dependent growth of cancer cells (estrogen antagonist). BCA and GEN bind preferentially to ERβ compared with ERα (41). ERα expression is high in MCF7 cells (42) but low in HMEC (43) and very low in MCF12A cells (44); on the other hand, ERβ is expressed in high levels in HMEC (43) and very low levels in MCF12A and MCF7 cells (42,44). In our study, the effects of BCA appear not to be related to ER expression, whereas the undesirable effects of GEN with regard to cancer prevention may be correlated with the ERβ expression level (Table 5). Furthermore, GEN can up-regulate ERβ (45), which could then produce greater effects in HMEC cells where ERβ is highly expressed (43). The potency and efficacy of BCA and GEN on binding to ER may also contribute to their different effects (46). BCA is considerably less likely to bind to ERs than GEN. This is attributed to the presence of the methoxy group (instead of hydroxyl group). The difference in binding affinities to ER suggests that GEN and BCA may have different biological activities in cancer cells (46). Transactivation induced by GEN through ER was higher than by BCA (46,47). Thus, this might provide an explanation why there were more alterations of gene expression by GEN than BCA in our study. Metabolism of isoflavones may also be a factor explaining different effects of BCA and GEN on ER. Human breast can-
We investigated whether the effects of the isoflavones on gene expression are related to differing metabolism in different cell lines by determining concentrations at 48 h. Because of the extensive metabolism in MCF7 cells, additional experiments were performed at earlier time points (20 min and 1 h) in MCF7 cells (unpublished data). Determination of the metabolism of isoflavones at the target site is important because the parent compound or the formed metabolites may have biological activity. Because BCA is demethylated to GEN, it was of interest to determine if similar metabolite profiles are present following BCA and GEN exposure. We found higher levels of parent BCA than the metabolite GEN in all three types of mammary cells following BCA exposure. Different degrees of metabolism were observed in the three different cells; MCF7 cells extensively metabolized both isoflavones, whereas less metabolism was observed in MCF12A and HME cells. The large extent of metabolism of BCA in MCF7 cells may represent the reason that BCA had no effect on gene expression in MCF7 cells. An interesting finding in this investigation is that, in GENtreated cells, conjugated BCA was observed. Over time, levels of GEN conjugates decreased (85.3 ng/ml/mg protein at 20 min, 76.4 ng/ml/mg protein at 1 h, and 47.0 ng/ml/mg protein at 48 h), whereas BCA conjugates increased (26.8 ng/ml/mg protein at 20 min, 37.0 ng/ml/mg protein at 1 h, and 75.0 ng/ml/mg protein at 48 h, all expressed in terms of BCA). In HMEC and MCF12A cells, BCA conjugates were also observed after GEN treatment. These findings suggest that methylation occurred in all three types of cells.
Table 5. Summary of Altered Gene Expression by GEN and BCA With Respect to Beneficial and Undesirable Effects for Cancer Preventiona
BCA beneficial BCA undesirable GEN beneficial GEN undesirable
HMEC
MCF12A
MCF7
3 (p18, ATF-2, WT1) 0 6 (egr-1, gadd45, hsf-1, hsp27, IRF-1, PTEN) 11 (ATF-2, bax, DPC4, hsp90, mdm2, p19, p21, p53, pig8, VHL)
4 (CD5, IκBα, MSH2, NF2) 1 (p27) 6 (CD5, NF2, p18, p19, PTEN, Rb) 0
0 0 4 (p21, p53, pig8, PTEN) 2 (APC, BRCA1)
a : GEN, genistein; BCA, biochanin A.
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In BCA-treated cells, the same metabolic patterns were observed at 48 h and at earlier time points (20 min and 1 h): BCA conjugates > parent BCA > GEN conjugates > metabolite GEN. GEN-treated MCF7 cells also exhibited similar patterns of metabolism when evaluated at 48 h, 20 min, and 1 h: GEN conjugates > parent GEN > BCA conjugates, and no metabolite BCA was observed (data not shown). Conclusion In conclusion, the effects of BCA and GEN treatment on gene expression in normal mammary cells of finite lifespan, a nontumorigenic human mammary epithelial cell line (MCF12A), and human breast cancer cells (MCF-7) differed. BCA treatment produced beneficial chemopreventive alterations in gene expression, whereas GEN showed mostly undesirable effects in normal HMEC cells, at concentrations that can be obtained following dietary intake. This suggests that BCA may represent a better breast cancer–preventive agent than GEN. However, the overall effect elicited by beneficial and adverse transcriptional changes needs further study. The use of HMEC cells may be useful as a first step in understanding the mechanisms of breast cancer prevention produced by isoflavones as well as other compounds. Acknowledgments and Notes We thank Dr. Kazuko Sagawa of Pfizer for performing the LC/MS/MS assay. This work was supported by grants from the Susan G. Komen Foundation, Cancer Research and Prevention Foundation, and Kapoor Charitable Foundation, University at Buffalo. Address correspondence to M. E. Morris, Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State University of New York, Amherst, NY 14260. Phone: 716– 645–2842 (X230). FAX: 716–645–3693. E-mail:
[email protected]. Submitted 19 April 2006; accepted in final form 21 September 2006.
References 1. Zafonte BT, Hulit J, Amanatullah DF, Albanese C, Wang C, et al.: Cellcycle dysregulation in breast cancer: breast cancer therapies targeting the cell cycle. Front Biosci 5, D938–D961, 2000. 2. Lamartiniere CA: Protection against breast cancer with genistein: a component of soy. Am J Clin Nutr 71, S1705–S1707; discussion S1708– S1709, 2000. 3. Barnes S, Peterson G, Grubbs C, and Setchell K: Potential role of dietary isoflavones in the prevention of cancer. Adv Exp Med Biol 354, 135–147, 1994. 4. Sarkar FH and Li Y: Soy isoflavones and cancer prevention. Cancer Invest 21, 744–757, 2003. 5. Wu Q, Wang M, and Simon JE: Determination of isoflavones in red clover and related species by high-performance liquid chromatography combined with ultraviolet and mass spectrometric detection. J Chromatogr A 1016, 195–209, 2003. 6. Ying C, Hsu JT, Hung HC, Lin DH, Chen LF, et al.: Growth and cell cycle regulation by isoflavones in human breast carcinoma cells. Reprod Nutr Dev 42, 55–64, 2002. 7. Ying C, Hsu JT, and Shieh SC: Growth inhibition of human endothelial cells by the phyto-oestrogen biochanin A, a metabolite of genistein. Br J Nutr 85, 615–620, 2001.
Vol. 57, No. 1
8. Mizunuma H, Kanazawa K, Ogura S, Otsuka S, and Nagai H: Anticarcinogenic effects of isoflavones may be mediated by genistein in mouse mammary tumor virus-induced breast cancer. Oncology 62, 78– 84, 2002. 9. Peterson TG, Ji GP, Kirk M, Coward L, Falany CN, et al.: Metabolism of the isoflavones genistein and biochanin A in human breast cancer cell lines. Am J Clin Nutr 68, S1505–S1511, 1998. 10. Peterson TG, Coward L, Kirk M, Falany CN, and Barnes S: The role of metabolism in mammary epithelial cell growth inhibition by the isoflavones genistein and biochanin A. Carcinogenesis 17, 1861–1869, 1996. 11. Pike AC, Brzozowski AM, Hubbard RE, Bonn T, Thorsell AG, et al.: Structure of the ligand-binding domain of oestrogen receptor beta in the presence of a partial agonist and a full antagonist. EMBO J 18, 4608–4618, 1999. 12. Allred CD, Allred KF, Ju YH, Virant SM, and Helferich WG: Soy diets containing varying amounts of genistein stimulate growth of estrogendependent (MCF-7) tumors in a dose-dependent manner. Cancer Res 61, 5045–5050, 2001. 13. Barnes S: Phyto-oestrogens and osteoporosis: what is a safe dose? Br J Nutr 89 (suppl 1), S101–S108, 2003. 14. Bartley JC and Stampfer MR: Factors influencing benzo[a]pyrene metabolism in human mammary epithelial cells in culture. Carcinogenesis 6, 1017–1022, 1985. 15. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, et al.: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3, research0034.1–research0034.11, 2002. 16. Loeb DM, Korz D, Katsnelson M, Burwell EA, Friedman AD, et al.: Cyclin E is a target of WT1 transcriptional repression. J Biol Chem 277, 19627–19632, 2002. 17. Idelman G, Glaser T, Roberts CT Jr, and Werner H: WT1-p53 interactions in insulin-like growth factor-I receptor gene regulation. J Biol Chem 278, 3474–3482, 2003. 18. Van Antwerp DJ, Martin SJ, Kafri T, Green DR, and Verma IM: Suppression of TNF-alpha-induced apoptosis by NF-kappaB. Science 274, 787–789, 1996. 19. Papaconstantinou AD, Goering PL, Umbreit TH, and Brown KM: Regulation of uterine hsp90alpha, hsp72 and HSF-1 transcription in B6C3F1 mice by beta-estradiol and bisphenol A: involvement of the estrogen receptor and protein kinase C. Toxicol Lett 144, 257–270, 2003. 20. Parcellier A, Schmitt E, Gurbuxani S, Seigneurin-Berny D, Pance A, et al.: HSP27 is a ubiquitin-binding protein involved in I-kappaBalpha proteasomal degradation. Mol Cell Biol 23, 5790–5802, 2003. 21. Scharnhorst V, Menke AL, Attema J, Haneveld JK, Riteco N, et al.: EGR-1 enhances tumor growth and modulates the effect of the Wilms’ tumor 1 gene products on tumorigenicity. Oncogene 19, 791–800, 2000. 22. Del-Val M and Lopez D: Multiple proteases process viral antigens for presentation by MHC class I molecules to CD8(+) T lymphocytes. Mol Immunol 39, 235–247, 2002. 23. Bianchi AB, Hara T, Ramesh V, Gao J, Klein-Szanto AJ, et al.: Mutations in transcript isoforms of the neurofibromatosis 2 gene in multiple human tumour types. Nat Genet 6, 185–192, 1994. 24. Woo IS, Kohno T, Inoue K, Ishii S, and Yokota J: Infrequent mutations of the activating transcription factor-2 gene in human lung cancer, neuroblastoma and breast cancer. Int J Oncol 20, 527–531, 2002. 25. Singhal S, Wiewrodt R, Malden LD, Amin KM, Matzie K, et al.: Gene expression profiling of malignant mesothelioma. Clin Cancer Res 9, 3080–3097, 2003. 26. Ahr A, Holtrich U, Solbach C, Scharl A, Strebhardt K, et al.: Molecular classification of breast cancer patients by gene expression profiling. J Pathol 195, 312–320, 2001. 27. Kobayashi D, Yamada M, Kamagata C, Kaneko R, Tsuji N, et al.: Overexpression of early growth response-1 as a metastasis-regulatory factor in gastric cancer. Anticancer Res 22, 3963–3970, 2002. 28. Fahmy RG, Dass CR, Sun LQ, Chesterman CN, and Khachigian LM: Transcription factor Egr-1 supports FGF-dependent angiogenesis
57
29.
30.
31.
32.
33.
34. 35.
36.
37. 38.
39.
40.
58
during neovascularization and tumor growth. Nat Med 9, 1026–1032, 2003. Dave B, Eason RR, Till SR, Geng Y, Velarde MC, et al.: The soy isoflavone genistein promotes apoptosis in mammary epithelial cells by inducing the tumor suppressor PTEN. Carcinogenesis 26, 1793– 1803, 2005. Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, et al.: Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 15, 6541–6551, 1996. Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, et al.: Regulation of cell death protease caspase-9 by phosphorylation. Science 282, 1318–1321, 1998. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, et al.: Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857–868, 1999. Cantley LC and Neel BG: New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3kinase/AKT pathway. Proc Natl Acad Sci USA 96, 4240–4245, 1999. Waite KA, Sinden MR, and Eng C: Phytoestrogen exposure elevates PTEN levels. Hum Mol Genet 14, 1457–1463, 2005. Weng LP, Brown JL, and Eng C: PTEN coordinates G(1) arrest by down-regulating cyclin D1 via its protein phosphatase activity and up-regulating p27 via its lipid phosphatase activity in a breast cancer model. Hum Mol Genet 10, 599–604, 2001. Foster JS, Henley DC, Ahamed S, and Wimalasena J: Estrogens and cell-cycle regulation in breast cancer. Trends Endocrinol Metab 12, 320–327, 2001. Sarkar FH and Li Y: Mechanisms of cancer chemoprevention by soy isoflavone genistein. Cancer Metastasis Rev 21, 265–280, 2002. de Jager T, Pelzer T, Muller-Botz S, Imam A, Muck J, et al.: Mechanisms of estrogen receptor action in the myocardium. Rapid gene activation via the ERK1/2 pathway and serum response elements. J Biol Chem 276, 27873–27880, 2001. Pratt MA, Satkunaratnam A, and Novosad DM: Estrogen activates raf1 kinase and induces expression of Egr-1 in MCF-7 breast cancer cells. Mol Cell Biochem 189, 119–125, 1998. Foster JS, Fernando RI, Ishida N, Nakayama KI, and Wimalasena J: Estrogens down-regulate p27Kip1 in breast cancer cells through Skp2 and through nuclear export mediated by the ERK pathway. J Biol Chem 278, 41355–41366, 2003.
41. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, et al.: Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 138, 863–870, 1997. 42. Paruthiyil S, Parmar H, Kerekatte V, Cunha GR, Firestone GL, et al.: Estrogen receptor beta inhibits human breast cancer cell proliferation and tumor formation by causing a G2 cell cycle arrest. Cancer Res 64, 423–428, 2004. 43. Zhao C, Lam EW, Sunters A, Enmark E, De Bella MT, et al.: Expression of estrogen receptor beta isoforms in normal breast epithelial cells and breast cancer: regulation by methylation. Oncogene 22, 7600–7606, 2003. 44. Zelinski DP, Zantek ND, Walker-Daniels J, Peters MA, Taparowsky EJ, et al.: Estrogen and Myc negatively regulate expression of the EphA2 tyrosine kinase. J Cell Biochem 85, 714–720, 2002. 45. Power KA and Thompson LU: Ligand-induced regulation of ERalpha and ERbeta is indicative of human breast cancer cell proliferation. Breast Cancer Res Treat 81, 209–221, 2003. 46. Beck V, Unterrieder E, Krenn L, Kubelka W, and Jungbauer A: Comparison of hormonal activity (estrogen, androgen and progestin) of standardized plant extracts for large scale use in hormone replacement therapy. J Steroid Biochem Mol Biol 84, 259–268, 2003. 47. An J, Tzagarakis-Foster C, Scharschmidt TC, Lomri N, and Leitman DC: Estrogen receptor beta-selective transcriptional activity and recruitment of coregulators by phytoestrogens. J Biol Chem 276, 17808– 17814, 2001. 48. Peng B, Lu B, Leygue E, and Murphy LC: Putative functional characteristics of human estrogen receptor-beta isoforms. J Mol Endocrinol 30, 13–29, 2003. 49. Boersma BJ, Patel RP, Kirk M, Jackson PL, Muccio D, et al.: Chlorination and nitration of soy isoflavones. Arch Biochem Biophys 368, 265–275, 1999. 50. Vladusic EA, Hornby AE, Guerra-Vladusic FK, Lakins J, and Lupu R: Expression and regulation of estrogen receptor beta in human breast tumors and cell lines. Oncol Rep 7, 157–167, 2000. 51. Vissac-Sabatier C, Bignon YJ, and Bernard-Gallon DJ: Effects of the phytoestrogens genistein and daidzein on BRCA2 tumor suppressor gene expression in breast cell lines. Nutr Cancer 45, 247–255, 2003.
Nutrition and Cancer 2007
