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Nutrition and Disease

Soy Isoflavones Alter Expression of Genes Associated with Cancer Progression, Including Interleukin-8, in Androgen-Independent PC-3 Human Prostate Cancer Cells¹

Renita Handayani^*,2, Lori Rice^,2,3, Yuehua Cui^**, Theresa A. Medrano^*, Von G. Samedi^*, Henry V. Baker, Nancy J. Szabo and Kathleen T. Shiverick^*

Departments of ^* Pharmacology and Therapeutics, Surgery, Molecular Genetics and Microbiology, College of Medicine, Analytical Toxicology Core Laboratory, College of Veterinary Medicine, and ^** Department of Statistics, College of Liberal Arts and Sciences, University of Florida, Gainesville, FL 32610

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

ABSTRACT

High consumption of soy isoflavones in Asian diets has been correlated with a lower incidence of clinically important cases of prostate cancer. The chemopreventive properties of these diets may result from an interaction of several types of isoflavones, including genistein and daidzein. The present study investigated the effects of a soy isoflavone concentrate (ISF) on growth and gene expression profiles of PC-3 human prostate cancer cells. Trypan blue exclusion and [³H]-thymidine incorporation assays showed that ISF decreased cell viability and caused a dose-dependent inhibition of DNA synthesis, respectively, with 50% inhibition (IC₅₀) of DNA synthesis at 52 mg/L (P = 0.05). The glucoside conjugates of genistein and daidzein in ISF were converted to bioactive free aglycones in cell culture in association with the inhibition of DNA synthesis. Flow cytometry and Western immunoblot analyses showed that ISF at 200 mg/L caused an accumulation of cells in the G₂/M phase of the cell cycle (P < 0.05) and decreased cyclin A by 20% (P < 0.05), respectively. The effect of ISF on the gene expression profile of PC-3 cells was analyzed using Affymetrix oligonucleotide DNA microarrays that interrogate 17,000 human genes. Of the 75 genes altered by ISF, 28 were upregulated and 47 were downregulated (P < 0.05). Further analysis showed that IL-8, matrix metalloproteinase 13, inhibin ß A, follistatin, and fibronectin mRNA levels were significantly reduced, whereas the expression of p21^CIP1, a major cell cycle inhibitory protein, was increased. The effects of ISF on the expression of IL-8 and p21^CIP1 mRNA and protein were validated at high and low ISF concentrations. Our data show that ISF inhibits the growth of PC-3 cells through modulation of cell cycle progression and the expression of genes involved in cell cycle regulation, metastasis, and angiogenesis.

KEY WORDS: • interleukin-8 • isoflavones • microarray • p21^CIP1 • prostate

In 2005, prostate cancer will be the most commonly diagnosed malignancy in the United States, with an estimated 232,000 new cases, and the second leading cause of cancer mortality in men, with >30,000 deaths expected (1). Similar statistics are found in other Western countries. However, epidemiologic studies point to lower incidence and mortality rates in Asian countries (2). Consumption of foods rich in soy isoflavones has been associated with a reduced risk of prostate cancer (2–4). Moreover, soy isoflavones were shown to protect cells from oxidative stress–inducing agents by inhibiting nuclear factor-B activation and decreasing DNA adduct levels in healthy volunteers (5).

Soy isoflavone extracts were shown to significantly inhibit prostate cancer cell growth in vitro, as well as tumor growth in mice (6,7). Genistein, the predominant isoflavone in soy, was shown to have antioxidant activity and to inhibit tumor growth through antiproliferative and antiangiogenic mechanisms (6,7). We reported previously that biochanin A, an isoflavone found in red clover, inhibited the growth of LNCaP prostate cancer cells through induction of cell cycle arrest and apoptosis (8).

The present study investigated the effects of a soy isoflavone concentrate (ISF)⁴ on the growth and gene expression profiles in androgen-independent, p53 null, PC-3 cells, a model of advanced prostate cancer. A similar ISF concentrate was shown to significantly inhibit prostate tumor growth in mice (6,7,9). In our study, ISF-regulated genes were identified using oligonucleotide Affymetrix U133A GeneChips that probe 17,000 human genes. Genes of interest upregulated by ISF include p21^CIP1, a major cyclin-dependent kinase (cdk) inhibitor (10), whereas downregulated genes were involved in cell invasion, metastasis, and angiogenesis, including IL-8, a potent chemokine and angiogenic factor. The hypothesis that isoflavones downregulate IL-8 is attractive because of recent evidence linking it to the stimulation of angiogenesis, tumorigenicity, and metastatic potential in several types of cancer cells, including prostate (11–14). IL-8 expression has been directly associated with Gleason score and tumor grade (12), with higher circulating levels found in men with metastatic lesions than in those with localized disease (13).

MATERIALS AND METHODS

    Cell culture and treatments. The human prostate carcinoma PC-3cell line, obtained from American Type Cell Culture Collection was routinely maintained in F12K HAM medium (Sigma-Aldrich), supplemented with 10% fetal bovine serum, 100 kU/L penicillin-streptomycin and 2 mmol/L L-glutamine. Cells were grown in a humidified 5% CO₂ incubator at 37°C. NovaSoy® is a soy phytochemical concentrate and commercially available dietary supplement provided by Archer Daniels Midland Company. This ISF product is prepared through an ethanol extraction process (6) and contains 49% isoflavones by weight. NovaSoy approximates the natural composition of isoflavones in soybeans with a low content of soy protein (8.5%) and fat (0.42%). In our experiments, NovaSoy was dissolved in dimethyl sulfoxide (DMSO) and PC-3 cells were treated with varying ISF concentrations for 48 h. Control cultures were treated with DMSO vehicle alone with a final concentration of 0.1%.

    Isoflavone analysis. Concentrations of free and conjugated glucosides in ISF-treated culture media were measured using reversed-phase HPLC with UV and MS detection in series (15,16). Aliquots of media samples were analyzed directly for free aglycone genistein and daidzein, and another aliquot underwent enzymatic hydrolysis with ß-glucosidase (Sigma) to determine total genistein and daidzein. Briefly, samples were prepared for analysis by mixing with ammonium acetate buffer and formic acid before multiple extractions in ethyl acetate. Pooled extracts were suspended in 50:50 acetonitrile:0.2% aqueous formic acid and injected onto an Apollo C18 column with Alltech Adsorbosphere HS C18 guard column in a Hewlett-Packard 1100 series liquid chromatograph system. Separation occurred under a linear gradient with mobile phase B (0.1% formic acid in acetonitrile) increasing from 40 to 60% over 60 min. Mobile phase A was 0.1% formic acid in water with a flow rate of 0.40 mL/min and column temperature of 40°C. Free isoflavones and deconjugated glucosides were detected for quantitation using an HP variable wavelength UV detector at 260 nm and quantified against an external standard series. Identification was confirmed using the Finnigan LCQ Ion Trap Mass Spectrometer in positive ion mode with electrospray ionization. Extraction efficiency was determined using the isoflavone biochanin A, with phenolphthalein-glucuronide as deconjugation surrogate.

    Cell viability. PC-3 cell viability was determined by the ability of cells to exclude 0.1% trypan blue. Cell viability was expressed as the total number of cells able to exclude dye relative to the controls.

    [³H]-thymidine incorporation assay. Cells were treated with 0.1–200 mg/L ISF or DMSO for 48 h. [³H]-thymidine (4 µCi/well) was then added to the medium and pulsed for 16 h. Cells were collected on glass fiber filters using a Brandel cell harvester and the amount of incorporated [³H]-thymidine was determined by liquid scintillation counting.

    Flow cytometry. Isolation and staining of cell nuclei was performed using the Cycle TEST^TM PLUS DNA reagent kit (Becton Dickinson) according to the manufacturer's protocol. Briefly, cells were treated with a nonionic detergent and trypsin to digest cell membranes. The nuclei were then treated with trypsin inhibitor, RNase, and propidium iodide to label the DNA. The cells were then subjected to flow cytometric analysis on FACSort (Becton Dickinson) and analyzed using the CELL Fit software program.

    Western immunoblots. Cell lysates (30 µg protein) were analyzed by SDS-PAGE as described previously (8). Briefly, after blocking, the membranes were incubated with anti-human, cyclin A, cyclin B, or p21^CIP1 mouse monoclonal antibodies (BD Transduction Laboratories), or with anti-human glutathione S-transferase (GST)-A1, -M1, and -P1 polyclonal rabbit antibodies (Oxford Biomedical Research). Proteins were detected with horseradish peroxidase-conjugated anti-mouse or -rabbit IgG antibody using enhanced chemiluminescence detection system (Amersham Pharmacia Biotech UK). Anti-human actin mouse monoclonal antibody (Oncogene Research Products) was used to verify equal loading and transfer efficiency.

    Northern blots. Total RNA was isolated using the guanidine-thiocyanate method (17), followed by formaldehyde-agarose gel electrophoresis and transfer onto a Magnacharge nylon membrane (Micron Separations). After UV-crosslinking, membranes were hybridized with a ³²P-labeled probe prepared from a plasmid containing p21 cDNA obtained from Dr. Bert Vogelstein (Johns Hopkins Oncology center, Baltimore, MD) (18). Membranes were exposed to radiographic film for detection of mRNA signals and quantitated with Scion Image software. The signals were normalized for the total RNA loading and transfer efficiency with ß-actin mRNA.

    Real-time PCR. Total RNA was reverse transcribed to cDNA using Taqman® Reverse Transcription Reagents (Applied Biosystems). Cytokine mRNA expression was determined in duplicate by real-time PCR on a multiplex Taqman Cytokine Gene Expression Plate 1 (Applied Biosystems), using 18S ribosomal RNA endogenous control. cDNA was amplified using the ABI Prism 7700 Sequence Detection System, with results given as the cycle number (C_T) at which a cDNA transcript reaches a selected amplification threshold. Data were expressed by normalizing the expression (in C_T) of each cytokine against the expression of the 18S control in which the C_T for a given transcript is inversely proportional to the level of that transcript in the original sample. By normalizing the expression of each cytokine against the 18S control, cytokine cDNA levels in different samples could be compared quantitatively between plates.

    ELISA. PC-3 cells were seeded into 6-well plates and treated with ISF at 200 mg/L for 48 h. Culture media from the plates were assayed using Quantikine human IL-8 immunoassay kit (R&D Systems) according to the manufacturer's protocol.

    Statistical analysis of biological assays. Experiments were repeated 3 times, and all treatments were expressed relative to the control, which was set at 100%. Results from 3 experiments are presented as means ± SEM. Data were analyzed by ANOVA, using Tukey's post hoc test to determine pairwise comparisons. Statistical analyses were performed using Microsoft Excel with Analyze-It add-in software. Differences were considered significant at P 0.05.

Microarray chip processing and data analysis

    Gene expression profiling using oligonucleotide microarray chips. The expressed genomes of ISF-treated and control cells were analyzed using Affymetrix Human Genome U133A GeneChip® microarrays, according to the manufacturer's protocol. These chips contain probe sets to interrogate >17, 000 transcripts. This set design uses sequences selected from GenBank, dbEST, and RefSeq, created from the UniGene database (Build 133, April 20, 2001). Briefly, at least 4 µg of total RNA extract was reverse-transcribed to cDNA using reagents obtained from Invitrogen. Biotin-labeled cRNA samples were produced by in vitro transcription from the cDNA templates by incorporating biotinylated nucleotides using an ENZO BioArray HighYield^TM RNA Transcript Labeling Kit (T7) (Enzo Life Sciences) and subsequently hybridized onto the microarray chips. The GeneChips were then scanned with an Affymetrix array scanner, and the fluorescence intensity measurements calculated using Affymetrix Microarray Suite 5.0 (MAS 5.0).

    Microarray data processing. Microarray image files (.CEL) were first analyzed using Affymetrix Microarray Suite (MAS) 5.0 and present/absent calls were generated for each probe set in MAS. Then the image files were loaded onto DNA-Chip Analyzer (dChip) software (19) together with the text files that were generated in MAS 5.0 and contained the MAS present/absent calls. The default normalization method (Invariant Set Normalization) in dChip was applied and the model-based expression intensity and standard error (SE) were calculated for each probe set.

    Microarray data analysis. Hierarchical clustering was performed using Pearson correlation with average linkage as implemented in dChip. Genes were first filtered according to the following criteria: 1) CV (SD/mean) > 0.5, and 2) the percentage of present calls in the arrays used > 20%. Then filtered genes were checked for data quality using sample clustering to determine whether replicate samples clustered together and to identify unexpected clustering patterns (e.g., cluster showing batch effects). Normalized expression intensities were then visualized using colorimetric matrices with red colors indicating relative overexpression and green colors indicating relative underexpression for a given probe set. Any batch effect can be considered in later analysis. Accordingly, sample comparison is restricted within batches. The same clustering method was also applied to gene cluster. For a small number of interesting genes, average-linkage hierarchical clustering of the data was applied using Cluster and the results displayed using TreeView (20).

An unpaired 2-group comparison for all probe sets was performed. Genes were determined to have altered expression levels in ISF-treated samples (experiment) vs. DMSO control samples based on the following criteria: 1) P-value for 2-sample t test 0.05, and 2) 1.4-fold change in the expression levels between the means of the 2 groups. The lower confidence bound of the 90% CI of the fold changes was used. It was shown that the lower confidence bound is more reliable than fold change as a ranking statistic for changes in gene expression (21); 3) an absolute difference between the means of the expression levels of the 2 groups >50. The reliability of the comparison criteria was assessed by checking the false discovery rate via permutation of the samples. Genes that satisfied all of the above criteria were considered significantly different between conditions. Significantly affected genes were then categorized on the bases of their cellular component, biological process, and molecular function using Onto-Express (22).

RESULTS

    Effects of ISF on DNA synthesis and viability in PC-3 cells. ISF decreased cell viability and caused a dose-dependent inhibition of DNA synthesis in PC-3 cells (Fig. 1A). DNA synthesis was inhibited by 50% (IC₅₀) at 52 mg/L (P = 0.05) without any effect on viability. Doses <50 mg/L did not affect either variable. ISF at 200 mg/L inhibited DNA synthesis by 90% (IC₉₀) (P < 0.01), which we designated as a cytostatic concentration. Although doses of 100–200 mg/L reduced viability by 41% (P < 0.05) compared with controls or with cells treated with doses <50 mg/L, there was no evidence of activation of apoptosis on the basis of terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling DNA fragmentation assays.

View larger version (26K): [in this window] [in a new window]   FIGURE 1  Effects of soy isoflavones on DNA synthesis and cell viability in PC-3 cells compared with controls treated with DMSO vehicle alone. (A) Effects on DNA synthesis and cell viability in cells treated with 0.1–200 mg/L ISF. (B) Effects of 46 µmol/L genistein (GEN), 40 µmol/L daidzein (DAI), a combination of GEN+DAI, or 200 mg/L ISFs (75 µmol/L GEN; 45 µmol/L DAI) on DNA synthesis. Bars represent mean ± SEM, n = 3 experiments. Means without a common letter differ, P < 0.05. Asterisks indicate a difference from control: (A) *P < 0.05, **P < 0.01; (B) **P < 0.001.

    Effect of ISF on cell cycle progression. ISF increased the accumulation of cells in the G₂/M phase by 60% (P = 0.04) (DMSO 9.2 ± 2.3 vs. ISF 15.4 ± 0.6). Western immunoblots assaying the expression of cell cycle regulatory proteins showed that 200 mg/L ISF decreased the level of cyclin A by 20% (P < 0.05). In comparison, 200 mg/L ISF did not affect cyclin B in PC-3 cells.

    Effects of ISF on gene expression profiles. Statistical analysis using dChip software showed that 75 genes were altered by ISF treatment (P < 0.05) with 47 genes downregulated and 28 genes upregulated, as shown by a hierarchical cluster image produced by TreeView based on similar patterns of expression (Fig. 2A). Closer inspection of differentially expressed genes of interest (Fig. 2B) showed that ISF downregulated genes included IL-8, matrix metalloproteinase 13 (MMP-13), inhibin ß A, follistatin, and fibronectin; a number of these genes are linked to malignant progression. ISF upregulated genes including p21^CIP1, long-chain fatty acid CoA ligase 3, and NADPH dehydrogenase, quinone 1. Genes altered by ISF treatment were further analyzed according to ontology (Table 2) and were found to be involved in cell metabolism and growth, and molecular functions; they acted as transcription factors and influenced cell cytoskeleton, adhesion, and invasion pathways. Experiments were then performed to verify the alteration of gene expression profiles in ISF-treated PC-3 cells using real-time PCR, and Western and Northern blot analyses on selected genes, including p21^CIP1 and IL-8.

View larger version (55K): [in this window] [in a new window]   FIGURE 2  Hierarchical cluster images of genes significantly altered by ISF treatment in PC-3 cells generated by Cluster and TreeView. The color scale is obtained by normalization so that the magnitude (sum of the squares of the values) of a row vector is equal to 1. The treatment groups are DMSO, cells treated with 0.1% DMSO vehicle alone, and NS200, cells treated with ISF extract (NovaSoy). (A) Cluster image of 75 differentially expressed genes, selected using multiple comparison criteria; 47 genes were downregulated, and 28 genes upregulated. Y-axis brackets indicate clustering of similarly regulated genes at a concentration of 200 mg/L. (B) Cluster image of a subset of 23 ISF-regulated genes of interest, selected from the larger cluster.

View larger version (55K): [in this window] [in a new window]   FIGURE 3  Validation of microarray data for p21CIP1 and IL-8 expression in PC-3 cells treated with ISF compared with controls treated with DMSO vehicle alone. Effects of ISF at 50 and 200 mg/L on the expression of (A) p21CIP1 mRNA and (B) p21CIP1 protein in PC-3 cells compared with controls. Representative blots and graphs of (A) Northern and (B) Western analyses with data normalized to ß-actin. (C) Effects of 48 h of exposure to 200 mg/L ISF on IL-8 mRNA. The abundance of IL-8 mRNA transcripts was normalized to 18S ribosomal RNA. (D) Effects of 72 h of exposure to 50 mg/L ISF on IL-8 protein secreted in culture media. Bars represent means ± SEM, n = 3–4 experiments. Means without a common letter differ, P < 0.05. Asterisks indicate a difference from control: *P < 0.05, **P < 0.01.

    Expression of GST after ISF treatment. We detected strong expression of GST-P1 by PC-3 cells that was not affected by ISF treatment (data not shown). GST-A1 and -M1 proteins were below the level of detection. These results are in agreement with our microarray data showing that only the GST-P1 form was highly expressed in PC-3 cells, with no change due to ISF treatment.

DISCUSSION

The growing interest in soy as a dietary factor associated with a reduced risk of some cancers has stimulated investigations into the mechanisms of isoflavone action. The major objective of our study was to characterize alterations in global gene expression in PC-3 human prostate cancer cells after treatment with an ISF concentrate that reflects the natural isoflavone composition found in soybeans. For background, dietary consumption of a similar ISF concentrate significantly inhibited growth of prostate tumors in mice (6,7,9). Although conversion of the glucosides to free isoflavones in vivo normally occurs in the intestinal tract, we determined that PC-3 cells generated metabolically active free isoflavones in culture. Therefore, this type of dietary supplement can be used for in vitro assays to determine how these agents affect cancer cell proliferation.

A comparison of the effects of the ISF concentrate with purified isoflavones shows that genistein produces the greatest inhibition of DNA synthesis, although there is some contribution by the addition of daidzein. However, the ISF extract used in this study, with its combination of isoflavones and other components, was more potent; a 50% inhibition was achieved with a dose containing 16 µmol/L genistein compared with 46 µmol/L for pure genistein alone. This is in agreement with the reported in vitro sensitivity of the PC-3 cell line to genistein (6,23). Although humans consuming a high-soy diet usually have circulating isoflavone concentrations close to 0.2 µmol/L (24), consumption of a soy flour extract was found to produce plasma levels of 7 µmol/L (25). Moreover, prostate cancer patients given high doses (300–600 mg) of NovaSoy had circulating genistein concentrations of up to 27 µmol/L, with no evidence of genotoxicity (26). For comparison, 5 mg genistein/d in mice resulted in serum levels of 30 µmol/L, an amount sufficient to decrease primary tumor growth and enhance radiosensitivity of intraprostatic PC-3 xenografts (27). Thus, it is likely that isoflavone concentrates can be consumed in quantities sufficient to exert biological effects. At the same time, it is recognized that higher doses of isoflavones are required in cell culture to attain the same degree of growth inhibition of prostate cancer cells as seen with xenograft tumors in mice fed an ISF-supplemented diet (6,7,23,27). In this context, however, multiple changes induced by isoflavones may have a greater effect in the microenvironment of solid tumors.

The present study sought to identify pathways involved in ISF-induced growth inhibition using Affymetrix GeneChips, which probe >17,000 human genes. ISF altered the expression of 75 genes that are involved in metabolism, adhesion, metastasis, angiogenesis, cell growth, and transcriptional regulation in PC-3 cells (P < 0.05). In comparison, Li and Sarkar (28,29), using Affymetrix U95 arrays, recently reported that genistein significantly altered the expression of 832 genes in PC-3 cells, and selected genes were identified that are involved in transcription, translation, cell proliferation, angiogenesis, and metastasis. However, many of these genes differed from those in our ISF dataset, indicating that the interaction of multiple isoflavones may lead to inhibitory mechanisms that differ from that observed with genistein alone.

Of particular interest in our study was the ability of ISF to downregulate the expression of IL-8, a proinflammatory chemokine, potent angiogenic factor, and autocrine growth factor (30). This was confirmed by the markedly reduced concentrations of both mRNA and secreted protein. Moreover, ISF decreased IL-8 secretion from PC-3 cells at the lower concentration of 50 mg/L. This finding is important because IL-8 can stimulate angiogenesis, tumorigenicity, and the metastatic potential of several cancer cells including melanoma, lung, and prostate (11,30). In addition, expression of IL-8 was directly associated with the Gleason score and pathologic tumor stage and distinguished organ-confined from non-confined tumors (12). Greater expression levels of IL-8 were found in highly metastatic prostate cancer cell lines and in tissue samples from prostate cancer patients (13). Transfection with IL-8 antisense RNA markedly reduced the invasion of prostate cancer cells (14). We propose that inhibition of IL-8 expression by an ISF concentrate would have a greater effect within the tumor microenvironment via suppression of vascular growth and cell proliferation. Both effects were observed by Zhou et al. (6,7,9) in mouse prostate xenograft tumors in vivo with an ISF concentrate similar to NovaSoy. Thus, our study provides a mechanism for a greater in vivo antitumorigenic effect within the microenvironment of solid tumors.

A major gene of interest upregulated by ISF is CDKN1A, which encodes the p21^CIP1 protein, an important cdk inhibitor involved in the regulation of the cell cycle at both the G₀/G₁ and G₂/M phases (10,18). The identification of ISF-induced expression of p21^CIP1 mRNA by microarray analysis was subsequently confirmed at both the mRNA and protein levels, and further shown at the lower concentration of 50 mg/L. Given that PC-3 cells have no functional p53, induction of p21^CIP1by ISF must occur in a p53-independent manner (10,18). Interestingly, several other genes upregulated by ISF were implicated in cholecalciferol-induced antiproliferative effects in prostate cancer cells, including long-chain fatty acid-CoA ligase 3 (31) and paxillin (32). In addition, ISFs upregulated NAD(P)H dehydrogenase, quinone 1, which is thought to inhibit prostate cell transformation by detoxification of carcinogens (33).

Microarray analysis further implicated other ISF-downregulated genes in the mechanisms of antitumorigenic effects. NAD-dependent 15-hydroxy-prostaglandin dehydrogenase, a key enzyme in the prostaglandin and lipoxin pathways, was downregulated by ISF, which may have potent chemopreventive effects (34). ISF also decreased NAD(P)H NOX5, an enzyme that generates reactive oxygen species. Interestingly, inhibitors of NOX5, including antioxidants, reduced proliferation and increased apoptosis in DU-145 cells (35). Microarray data showed that ISF downregulated the expression of MMP-13, fibronectin, and integrin. MMP-13 is a putative marker of invasive prostate cancer (36), and genistein was found to decrease expression of several members of the MMP family, including MMP-13, in prostate cancer bone metastasis in mice (37). Osteonectin/SPARC, involved in the invasiveness of breast and prostate cancer cells in bone metastases (38), was also decreased by 33% of control by ISF. Greater expression of osteonectin is associated with malignant prostate cell lines and tissue, and may support the growth of bone metastatic lesions via increased expression of angiogenic factors (39). It was suggested that GST-P1 has protective effects against genomic damage mediated by carcinogens and is not expressed commonly in prostate cancer (40). We found that the microarray data were validated by Western immunoblotting; the GST-P1 form was highly expressed in PC-3 cells, but expression was not affected by ISF treatment.

In summary, ISFs may inhibit the growth of prostate cancer cells by downregulation of genes involved in cell proliferation, metastasis, and angiogenesis, while inducing major cell cycle inhibitors. The use of soy extract supplements, such as the one investigated in this study, offers a way to obtain higher doses of ISFs compared with traditional diets that may be difficult for Americans to consume. These supplements maintain the ISF ratios found in soybeans and may retain other minor components that have potent anticancer activity, such as saponins and lunasin. In addition, about one-third of the population will convert substantial amounts of daidzein to equol via intestinal microbial metabolism, a metabolite with a high affinity for the estrogen receptor ß (41). This may provide them with added prostate cancer risk reduction. However, further investigations are warranted to establish the relation of ISF-regulated genes with the molecular mechanisms involved in cell proliferation, angiogenesis, and metastasis.

ACKNOWLEDGMENTS

We acknowledge Dr. Burt Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD) for plasmids containing p21 ^CIP1 cDNA.

FOOTNOTES

¹ Supported by grant #CA91231 from the National Cancer Institute, National Institutes of Health, and P42 ES07375 from the National Institute of Environmental Health Sciences.

² Drs. Handayani and Rice contributed equally in this work.

⁴ Abbreviations used: cdk, cyclin-dependent kinase; C_T, cycle number; DMSO, dimethyl sulfoxide; GST, glutathione S-transferase; IC, inhibitory concentration; ISF, soy isoflavone concentrate; MMP-13, matrix metalloproteinase 13; p21^CIP1, p21 cyclin inhibitory protein 1; PSA, prostate-specific antigen

Manuscript received 11 May 2005. Initial review completed 9 June 2005. Revision accepted 27 September 2005.

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