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Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI
2To whom correspondence should be addressed. E-mail: fsarkar{at}med.wayne.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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B (NF-B) and Akt signaling pathways. To better understand the precise molecular mechanism(s) by which genistein exerts its effects on PC3 cells, we utilized cDNA microarray to interrogate 12,558 known genes to determine the gene expression profiles altered by genistein treatment. We found a total of 832 genes that showed a greater than twofold change after genistein treatment from two independent experiments with a high degree of concordance. Among these genes, 774 genes were down-regulated and 58 genes were up-regulated with genistein treatment. Cluster analysis showed nine different types of expression alternations. These genes were also subjected to cluster analysis according to their biological functions. We found that genistein regulated the expression of genes that are critically involved in the regulation of cell growth, cell cycle, apoptosis, cell signaling transduction, angiogenesis, tumor cell invasion and metastasis. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to confirm the results of cDNA microarray, and the results of RT-PCR were consistent with the microarray data. We conclude that genistein affected the expression of a large number of genes that are related to the control of cell survival and physiologic behaviors. The gene expression profiles provide comprehensive molecular mechanism(s) by which genistein exerts its pleiotropic effects on cancer cells. Genistein-induced regulation of these genes may be further exploited for devising chemopreventive and/or therapeutic strategies for prostate cancer.
KEY WORDS: • genistein • gene expression • microarray • prostate cancer cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). However, the incidence and mortality of prostate cancer are significantly lower in Asians than in Western populations. Soybean foods comprise a large portion of the Asian diet, and high consumption of soybean foods has been associated with reduced risk of prostate cancer (2
). Genistein is a major isoflavone in soybeans, which has been found to inhibit carcinogenesis in vitro and in vivo (2
,3
). Our previous studies have shown that genistein inhibits the growth of PC3 prostate cancer cells and induces apoptosis by inhibiting nuclear factor B (NF-B)3 and Akt signaling pathways (4
,5
). Moreover, we found that genistein inhibits the expression of c-erbB2 and MMP-9, which play important roles in tumor cell invasion and metastasis (6
), suggesting that genistein elicits pleiotropic effects on cancer cells.
An increased understanding of molecular biological properties of anticancer agents will lead to the development of mechanism-based chemopreventive and/or therapeutic strategies. The incidence and mortality of prostate cancer are so high in the United States that there is a tremendous need for the development of mechanism-based and targeted strategies for prevention and treatment of prostate cancer. cDNA microarray analysis permits the simultaneous and rapid analysis of the expression of tens of thousands of genes, and, in turn, provides an opportunity for determining the effects of anticancer agents on prostate cancer cells (7
). This technology will contribute to the more accurate development of therapeutic strategies and will help to determine the molecular mechanism(s) of action of chemopreventive and/or therapeutic agents. To better understand the precise molecular mechanism(s) by which genistein exerts its effects on PC3 prostate cancer cells, we utilized a cDNA microarray to interrogate the mRNA levels of 12,558 genes and to determine the gene expression profiles of PC3 prostate cancer cells treated with genistein.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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PC3 human prostate cancer cells (ATCC, Manassas, VA) were cultured in RPMI-1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin in a 5% CO2 atmosphere at 37°C. Genistein (Toronto Research Chemicals, North York, Canada) was dissolved in 0.1 mol/L Na2CO3 to make a 10 mmol/L stock solution for experiments. For growth inhibition, PC3 cells were treated with 5, 15, 30 and 50 µmol/L genistein or 0.5 mmol/L Na2CO3 (vehicle control) for 1–3 d. The cells were then incubated with MTT (0.5 g/L, Sigma, St. Louis, MO) at 37°C for 4 h and with dimethyl sulfoxide at room temperature for 1 h. The spectrophotometric absorbance of the samples was measured using ULTRA Multifunctional Microplate Reader (TECAN, Durham, NC) at 495 nm. The experiment was repeated three times and t tests were done to determine whether cell growth was inhibited by the treatments.
cDNA microarray analysis for gene expression profiles.
PC3 cells were treated with 50 µmol/L genistein or 0.5 mmol/L Na2CO3 (vehicle control) for 6, 36 and 72 h. The rationale for choosing these time points was to capture gene expression profiles of early response genes, genes that may be involved during the onset of growth inhibition and apoptotic processes, and finally genes that may be involved as an executioner for induction of apoptosis. Total RNA from each sample was isolated by Trizol (Invitrogen) and purified by RNeasy Mini Kit and RNase-free DNase Set (QIAGEN, Valencia, CA) according to the manufactures’ protocols. cDNA for each sample was synthesized by using Superscript cDNA Synthesis Kit (Invitrogen) using T7-(dT)24 primer instead of the oligo(dT) provided in the kit. The biotin-labeled cRNA was transcripted in vitro from cDNA using a BioArray HighYield RNA Transcript Labeling Kit (ENZO Biochem, New York, NY) and purified using an RNeasy Mini Kit. The purified cRNA was fragmented by incubation in fragmentation buffer (200 mmol/L Tris-acetate pH 8.1, 500 mmol/L KOAc, 150 mmol/L MgOAc) at 95°C for 35 min and chilled on ice. The fragmented labeled cRNA was applied to Human Genome U95 Array (Affymetrix, Santa Clara, CA), which contains 12,588 human gene cDNA probes, and hybridized to the probes in the array. After washing and staining, the arrays were scanned using a HP GeneArray Scanner (Hewlett-Packard, Palo Alto, CA). Two independent experiments were performed to verify the reproducibility of results.
Microarray data normalization and analysis.
The gene expression levels of samples were normalized and analyzed using Microarray Suite, MicroDB and Data Mining Tool software (Affymetrix). The absolute call (present, marginal, absent) and average difference of 12,558 gene expressions in a sample, and the absolute call difference, fold change and average difference of gene expressions between two or several samples were normalized and identified using this software. Average differences of gene expression between treated and untreated samples that were greater than twofold were compared by t test. Average-linkage hierarchical clustering of the data was performed using cluster analysis (8
) and the results were displayed by with TreeView (8
). The genes showing altered expression were also categorized on the basis of their location, cellular component, reported or suggested biochemical, biological and molecular functions using Onto-Express (9
). Genes that were not annotated or not easily classified were excluded from the functional cluster analysis.
Reverse transcription-polymerase chain reaction (RT-PCR) analysis of gene expression.
The total RNA prepared for microarray was also used for RT-PCR. Total RNA (2µg) from each sample was subjected to reverse transcription using a Superscript first strand cDNA synthesis kit (Invitrogen) according to the manufacturer’s protocol. PCR reactions were then carried out by mixing 2 µL of cDNA, 5 µL of 10X PCR buffer, 1–2 µL 50 mmol/L MgCl2, 1 µL of 10 mmol/L dNTP, 2 µL of 10 µmol/L specific gene primer pair, 2 µL of 10 µmol/L ß-actin or glyceraldehyde-3-phosphate dehydrogenase primer pair, 35.5 µL H2O, and 0.5 µL of 5000 U/L Platinum Taq polymerase (Invitrogen) and amplified for 30 cycles. The primers used in the PCR reaction are presented in Table 1
. Each cycle consisted of denaturing for 1 min at 94°C, annealing for 1 min at 60°C and polymerization for 2 min at 72°C. The PCR products were resolved by electrophoresis through a 3% agarose gel and stained with ethidium bromide. The optical densities of PCR products in the agarose gel were scanned with a Gel Doc 1000 image scanner (Bio-Rad, Hercules, CA), and quantified using Molecular Analyst software (Bio-Rad).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The MTT assay showed that the treatment of PC3 prostate cancer cells with genistein dose and time dependently inhibited cell proliferation (Fig. 1
), demonstrating the growth inhibitory effect of genistein. These results are consistent with our previous study (5
). This inhibition of cell proliferation could be due to altered regulation of gene expression by genistein.
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The gene expression profiles of PC3 cells treated with genistein were assessed using cDNA microarray. Two independent experiments showed the expression of 832 genes at the mRNA level with more than a twofold change after genistein treatment. Among these genes, 774 were down-regulated and 58 were up-regulated by genistein. Cluster analysis showed nine different types of expression alternations (Fig. 2
). Cluster 1 and cluster 9 included the genes showing typical gradual decreases and increases at the level of expression (Tables 2
and 3
). All alternations of gene expression in clusters 1 and 9 were significant (P < 0.05). The altered expressions of most genes occurred as early as after 6 h of genistein treatment and were significantly greater with longer treatment. These genes were also subjected to cluster analysis according to location and cellular components. The genes showing altered expression were located mostly on chromosomes 1, 19, 17, 2, 6 and 11, and were mainly responsible for the transcription and translation of components of the nucleus and integral plasma membrane proteins (Table 4
). After clustering based on biological function, we found that genistein down-regulated genes that are involved mainly in signal transduction, oncogenesis, cell proliferation, protein phosphorylation and transcription. On the other hand, genistein up-regulated genes that are related mainly to signal transduction, protein dephosphorylation, heat shock response, inactivation of mitogen-activated protein kinase (MAPK), apoptosis and cell cycle arrest (Table 4
). When classified by molecular function, genistein down-regulated genes responsible for RNA binding, transcription factors, protein kinases including NF-B-inducing kinase and MAP kinase kinase, apoptosis inhibitor and collagenase. It up-regulated tumor suppressors, proteinase inhibitors and CDK inhibitors (Table 4
). Genistein also affected specific genes critically involved in the regulation of cell cycle, apoptosis, angiogenesis, invasion, metastasis and cell signaling pathways as shown in Table 5
.
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To verify the alterations of gene expression at the mRNA level, which appeared on the microarray, we chose 26 genes with varying expression profiles for RT-PCR analysis. The results of RT-PCR analysis for these selected genes were in direct agreement with the microarray data (Fig. 3
). The same alternations of gene expression were observed by RT-PCR analysis, although the fold change in expression was not always the same using these two different analytical methods. These results, however, support the findings obtained from microarray experiments and suggest that genistein regulates the transcription of the genes that are involved in the physiologic processes of prostate cancer cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–13
). The alternations of gene expression profiles by several anticancer agents have also been reported (14
,15
). Genistein is a protein tyrosine kinase inhibitor (16
). Our previous studies, along with the studies reported by other investigators, have shown that genistein may exert multiple inhibitory effects on cancer cells through regulation of several cell signal transduction pathways (4
–6
,17
,18
). However, little is known about the gene expression profiles of prostate cancer cells after genistein treatment. In this study, we utilized the high throughput gene chip, which contains 12,558 known genes, to determine the alternation of gene expression profiles of PC3 prostate cancer cells exposed to genistein. Our results from cDNA microarray provided a genome-wide analysis of the cellular response to genistein treatment. Cellular responses to any antiproliferative agents involve modulations of complex pathways that ultimately determine whether a cell survives or dies. Cellular and molecular responses of PC3 cells to genistein are complex and are likely to be mediated by a variety of regulatory pathways. We found that the molecular response to genistein in PC3 prostate cancer cells involved inhibition or induction of genes that are related to biochemical, biological and regulatory processes in the cells. These genes have specific functions in signal transduction, protein phosphorylation and dephosphorylation, cell proliferation and cell cycle control, transcriptional and translational regulation, protein degradation and cellular metabolism. They also control cell growth, apoptosis, oncogenesis, angiogenesis, invasion and metastasis. These results demonstrated that genistein regulates important genes critically involved in cell survival, physiologic behavior and cancer progression and may therefore be responsible for inhibiting the progression of prostate cancers. The alternations in the expression of these genes were observed at 6 h after genistein treatment, and continued to increase for 36 or 72 h, exhibiting time dependency. For example, the expression of urokinase plasminogen activator receptor (uPAR), protease M and bone-derived growth factor (BPGF) declined progressively until 72 h, whereas the expression of lamin B receptor, elafin, and vimentin increased progressively. The inhibition of cyclin B expression was relatively lower after 6 h of genistein treatment, and maximum inhibition occurred after 36–72 h. These results are consistent with the degree of cell growth inhibition as documented by 45 and 69.6% inhibition after 48 and 72 h of genistein treatment, respectively. These results suggest that genistein may modulate the expression of first-response genes at an earlier stage (6 h), and in turn, alter the expression of intracellular second messenger molecules, resulting in inhibition of PC3 prostate cancer cell growth and progression. The genes showing altered expression by genistein treatment reside mainly on chromosomes 17 and 19 where many critical genes for cell survival are located, and are mainly responsible for the transcription of nuclear and plasma membrane components, suggesting that genistein regulates the expression of important genes in cellular signaling.
When we categorized the genes showing alternations after genistein treatment according to their known function, we found that genistein affected the expression of several genes that are involved in tumor invasion and angiogenesis. Among these genes, type IV collagenase (MMP-9), urokinase plasminogen activator (uPA) and its receptor (uPAR), protease M, protease activated receptors (PAR-2), connective tissue growth factor and connective tissue activation peptide are very important genes in tumor cell invasion and metastasis (19
–23
). It has been reported that the cancer cells with higher levels of MMP-9, uPA or uPAR tend to invade surrounding tissues and subsequently migrate to blood vessels, thereby developing cancer cell metastasis (19
,20
). Connective tissue growth factor and connective tissue activation peptide promote the synthesis of the extracellular matrix and rebuild the structure of the extracellular matrix, inhibiting invasion and metastasis of cancer (23
). Vascular endothelial growth factor (VEGF), VEGF 165 receptor/neuropilin, transforming growth factor (TGF)-ß, thrombospondin (TSP), bone-derived growth factor (BPGF), and lysophosphatidic acid (LPA) play important roles in angiogenesis (24
–29
). Genistein down-regulated the expressions of MMP-9, uPA, uPAR, protease M, PAR-2, VEGF, VEGFR, TGF-ß, BPGF, LPA, and TSP, and up-regulated the expressions of connective tissue growth factor and connective tissue activation peptide, suggesting that it may inhibit angiogenesis, invasion and metastasis of PC3 prostate cancer cells.
From the gene expression profiles of PC3 cells exposed to genistein, we found that this compound inhibited the expression of seven genes (cyclin B, cyclin A, cdc25A, survivin, TGF-ß, ki67, pescadillo) that are involved in the regulation of the cell cycle and apoptosis. Four genes (p57KIP2, cyclin G2, growth arrest and DNA-damage-inducible protein, elafin) related to the control of cell growth were up-regulated. Cyclin B, cyclinA and cdc25A are cell cycle promoters, whereas p57KIP2 and cyclin G2 inhibit cell cycle progression (30
–32
). The inhibition of cyclin B expression shown by cDNA microarray is consistent with our earlier results showing the down-regulation of cyclin B in genistein-treated prostate cancer cells (33
). Survivin is an important inhibitor of apoptosis (34
). Pescadillo and ki67 promote cell growth, whereas growth arrest and DNA damage-inducible protein and elafin inhibit cell proliferation (35
–39
). Our results suggest that genistein may inhibit cell growth through regulation of the expression of these important genes related to cell proliferation. We also observed altered expression of genes encoding kinases including NF-B-inducing kinase (NIK) and MAP kinase kinase, differentiation factors, growth factors and transcription factors, suggesting that genistein inhibits the cell growth via mediation of cell signal transduction pathways. These results also provide mechanistic information in support of our previous observation that the inactivation of NF-B may be due to the inactivation of NIK (5
).
In summary, we have analyzed the gene expression profiles of PC3 prostate cancer cells exposed to genistein. Genistein altered the expressions of many genes that are related to the control of cell cycle, apoptosis, cell signaling transduction, angiogenesis, tumor cell invasion and metastasis, suggesting pleiotropic effects of this compound. The gene expression profiles revealed novel molecular mechanism(s) by which genistein exerts its inhibitory effects on prostate cancer cells. Genistein-induced regulation of these genes may be exploited for devising mechanism-based chemopreventive or therapeutic strategies for prostate cancer. However, further in-depth studies are required to investigate the effects of genistein on the regulation of important cellular molecules at the protein levels to examine the effects of genistein with cellular functions.
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FOOTNOTES |
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3 Abbreviations used: BPGF, bone-derived growth factor; LPA, lysophosphatidic acid; MAPK, mitogen-activated protein kinase; NF-B, nuclear factor B; NIK, NF-B-inducing kinase; PAR-2, protease activated receptors; RT-PCR, reverse transcription-polymerase chain reaction; TGF, transforming growth factor; TSP, thrombospondin; uPAR, urokinase plasminogen activator receptor; VEGF, vascular endothelial growth factor.
Manuscript received 20 July 2002. Initial review completed 16 August 2002. Revision accepted 10 September 2002.
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