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Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201
2To whom correspondence should be addressed. E-mail: fsarkar{at}med.wayne.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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B and Akt pathways. To obtain comprehensive gene expression profiles altered by I3C- and DIM-treated PC3 cells, we utilized cDNA microarray to interrogate the expression of 22,215 known genes using the Affymetrix Human Genome U133A Array. We found a total of 738 genes that showed a greater than twofold change after 24 h of DIM treatment. Among these genes, 677 genes were down-regulated and 61 were up-regulated. Similarly, 727 genes showed a greater than twofold change in expression, with down-regulation of 685 genes and up-regulation of 42 genes in I3C-treated cells. The altered expressions of genes were observed as early as 6 h and were more evident with longer treatment. Upon cluster analysis, we found that both I3C and DIM up-regulated the expression of genes that are related to the Phase I and Phase II enzymes, suggesting their increased capacity for detoxification of carcinogens or chemicals. We also found that I3C and DIM down-regulated the expression of genes that are critically involved in the regulation of cell growth, cell cycle, apoptosis, signal transduction, Pol II transcription factor and oncogenesis. Real-time reverse transcription-polymerase chain reaction analysis was conducted to confirm the cDNA microarray data, and the results were consistent. We conclude that I3C and DIM affected the expression of a large number of genes that are related to the control of carcinogenesis, cell survival and physiologic behaviors. This may help determine the molecular mechanism(s) by which I3C and DIM exert their pleiotropic effects on PC3 prostate cancer cells; in addition, this information could be further exploited for devising chemopreventive and/or therapeutic strategies for prostate cancer.
KEY WORDS: • indole-3-carbinol • 3,3'-diindolylmethane • gene expression • microarray • prostate cancer cells
Epidemiologic studies have shown that a high dietary intake of fruits and vegetables protects against carcinogenesis in many tissues (1 ,2 ). Among vegetables with anticarcinogenic properties, the cruciferous vegetable family including broccoli, cabbage, brussels sprouts and cauliflower appears to be most effective at reducing the risk of cancers (3 ,4 ). Indole-3-carbinol (I3C), a common phytochemical in the human diet, is present in almost all members of the cruciferous vegetable family. There is growing evidence showing that I3C has the potential to prevent or even treat a number of common cancers, especially those that are hormone-related (5 ,6 ). It has been reported that a diet rich in cruciferous vegetables or supplements of I3C caused regression of tumors or decreased the rate of growth in patients with recurrent laryngeal papillomatosis (7 ,8 ). The in vivo and in vitro studies have also demonstrated that I3C possesses anticarcinogenic effects in experimental animals and inhibits the growth of human cancer cells (9 ,10 ). Because of this information, interest in I3C as a cancer chemopreventive agent has increased greatly in the last few years.
I3C is chemically unstable in acidic environments and is rapidly converted in the stomach to a variety of condensation products. Among them, 3,3'-diindolylmethane (DIM) is a major acid condensation product of I3C in vitro and in vivo. Because of the ready conversion of I3C to DIM and other products under a variety of biological conditions, the biological effects of I3C may be attributable to both I3C and DIM. Experimental studies have revealed that DIM exhibits inhibitory effects on cancer cells by inhibiting cell growth and inducing apoptosis that are similar to the effects observed with I3C (11 ,12 ). It has been reported that DIM exerts its chemoprotective effects in estrogen-responsive tissues, and DIM-induced G1 arrest occurs with up-regulation of p21WAF1/CIP1 in breast cancer cells, suggesting its inhibitory effects on hormone-related cancers (11 ,13 ).
Prostate cancer is the most common nondermatological carcinoma in the United States with an estimated 189,000 new cases and 30,200 deaths in 2002 (14 ). Up to 30% of men undergoing radical prostatectomy will relapse, often as a result of micrometastatic disease present at the time of surgery (15 ). Thus, there is a tremendous need for the development of mechanism-based and targeted strategies for prevention and treatment of prostate cancer. cDNA microarray analysis allows us to examine the expression of tens of thousands of genes that can be monitored simultaneously and rapidly and, in turn, provides an opportunity to determine the effects of anticancer agents on prostate cancer cells (16 ). The gene expression profiles of various types of cancers were analyzed using cDNA microarray (17 ,18 ). The alterations of gene expression profiles by several anticancer agents have also been reported (19 ,20 ). This information is likely to contribute to devising preventive and/or therapeutic strategies more accurately, and will help to determine the molecular mechanism(s) of action of chemopreventive and/or therapeutic agents.
Our previous studies showed that I3C inhibits the growth of PC3 prostate cancer cells and induces apoptosis by inhibiting the nuclear factor (NF)-B and Akt signaling pathways, suggesting that I3C may serve as a preventive and/or therapeutic agent against prostate cancer (21
,22
). However, little is known about the global gene expression profiles of prostate cancer cells after I3C and DIM treatment; the precise molecular mechanism(s) by which I3C and DIM exert their tumor suppressive effects on prostate cancer is also unclear. In this study, we utilized the high throughput gene chip, which contains 22,215 known genes, to identify changes in gene expression in PC3 prostate cancer cells exposed to I3C and DIM.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS 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. I3C (Sigma, St. Louis, MO) or DIM (LKT, St. Paul, Minnesota) was dissolved in dimethyl sulfoxide (DMSO) to make a 100 or 50 mmol/L stock solution, respectively. For growth inhibition, PC3 cells were treated with 30, 60 and 100 µmol/L I3C or 15, 30, and 60 µmol/L DIM for 1–3 d. Control PC3 cells received 0.1% DMSO for the same time periods. The cells were then incubated with MTT (0.5 g/L, Sigma, St. Louis, MO) at 37°C for 4 h and with DMSO at room temperature for 1 h. The spectrophotometric absorbance of the samples was measured using the ULTRA Multifunctional Microplate Reader (TECAN, Durham, NC) at 495 nm. The experiment was repeated three times and t tests were performed to determine whether cell growth was inhibited by the treatments.
cDNA microarray analysis.
PC3 cells were treated with 60 µmol/L I3C or 40 µmol/L DIM for 6, 24 and 48 h. DIM is the in vivo dimeric product of I3C. The doses of I3C and DIM chosen for microarray experiment were close to the 50% inhibitory concentration. However, the biological relevance of these doses in relation to prevention or therapy has not been fully evaluated, although they may have therapeutic application. The rationale for choosing these time points was to capture the expression profiles of early-response genes, genes that may be involved in the onset of growth inhibition and apoptotic processes and, finally, genes that may act as performers for the induction of apoptosis. Total RNA from each sample was isolated by Trizol (Invitrogen, Carlsbad, CA) and purified using the RNeasy Mini Kit and RNase-free DNase Set (QIAGEN, Valencia, CA) according to the manufacture’s protocols. cDNA for each sample was synthesized using a Superscript cDNA Synthesis Kit (Invitrogen) and a T7-(dT)24 primer instead of the oligo(dT) provided in the kit. Then, the biotin-labeled cRNA was transcribed in vitro from cDNA using a BioArray HighYield RNA Transcript Labeling Kit (ENZO Biochem, New York, NY) and purified using the RNeasy Mini Kit. The purified cRNA was fragmented by incubation in fragmentation buffer (200 mmol/L Tris-acetate pH 8.1, 500 mmol/L potassium acetate, 150 mmol/L magnesium acetate) at 95°C for 35 min and chilled on ice. The fragmented labeled cRNA was applied to the Human Genome U133A Array (Affymetrix, Santa Clara, CA), which contains 22,215 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 signal value of the experimental array was multiplied by a normalization factor to make its mean intensity equivalent to the mean intensity of the control array using Microarray Suite software according to manufacturer’s protocol. The absolute call (present, marginal, absent) and average difference of 22,215 gene expressions in a sample, and the absolute call difference, fold change and average difference of gene expressions between two or several samples were identified using the above-mentioned software. Statistical analysis of the difference in the mean expression of genes that indicated a greater than twofold change was performed repeatedly between treated and untreated samples using t tests. Average-linkage hierarchical clustering of the data was applied using the Cluster (23 ) and the results were displayed with TreeView (23 ). The genes showing altered expression were also categorized on the basis of their location, cellular component and reported or suggested biochemical, biological and molecular functions using Onto-Express (24 ). Genes that were not annotated or not easily classified were excluded from the functional clustering analysis.
Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis.
The total RNA prepared for microarray analysis was also used for RT-PCR analysis of selected genes. Total RNA (2 µg) from each sample was subjected to reverse transcription using a Superscript first strand cDNA synthesis kit (Invitrogen) according to the manufacturer’s protocol. Real-time PCR reactions were then carried out in a 25 µL reaction mixture (2 µL of cDNA, 12.5 µL of 2X SYBR Green PCR Master Mix, 1.5 µL of 5 µmol/L specific gene primer pair and 9 µL of H2O) in an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). The sequences of primers used in the real-time PCR reaction were described previously (25 ). The PCR program was initiated by 2 min at 50°C and 10 min at 95°C before 40 thermal cycles, each of 15 s at 95°C and 1 min at 60°C. Data were analyzed according to the comparative cycle threshold (Ct) method and were normalized by actin expression in each sample. Melting curves for each PCR reaction were generated to ensure the purity of the amplification product.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The MTT assay showed that the treatment of PC3 prostate cancer cells with I3C or DIM resulted in dose- and time-dependent inhibitions of cell proliferation (Fig. 1 ), demonstrating the growth inhibitory effect of these compounds on PC3 cells. These results are consistent with our previously published results (22 ).
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The gene expression profiles of PC3 cells treated with I3C or DIM were assessed using cDNA microarray. Two independent experiments showed a total of 738 genes showing a greater than twofold change after 24 h of DIM treatment. Among these, 677 genes were down-regulated and 61 were up-regulated. Similarly, 727 genes showed a greater than twofold change in expression with down-regulation of 685 genes and up-regulation of 42 genes in I3C-treated cells. Clustering analysis showed 18 different types of expression alterations in DIM- or I3C-treated PC3 cells, respectively. Cluster 1 and cluster 18 included the genes showing typical gradual decreases and increases in expression, respectively. The altered expressions of genes occurred after only 6 h of I3C and DIM treatment, and were more evident with longer treatment (Fig. 2 ).
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To verify the alterations of gene expression at the mRNA level, which appeared on the microarray, we chose six genes [transforming growth factor-ß (TGF-ß)2, p57KIP2, cytochrome b5, eukaryotic protein synthesis initiation factor 4, thrombospondin 1 and neuropilin 1] with varying expression profiles for real-time RT-PCR analysis. The results of real-time RT-PCR analysis for these selected genes were consistent with the microarray data (Figs. 3 and 4 ). Gene expression alterations were similar by real-time RT-PCR analysis, although the fold changes in the expression level differed somewhat in the two analytical methods. These results support the findings obtained from microarray experiments, and also suggest that I3C and DIM regulate the transcription of genes that are involved in the physiologic processes of prostate cancer cells.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Because DIM is one of the major in vivo dimeric products of I3C, we also found that the molecular response to both I3C and DIM in PC3 prostate cancer cells involved mainly inhibition of genes that are related to cell growth, cell cycle control, apoptosis, signal transduction, oncogenesis, transcription regulation and protein phosphorylation in addition to the induction of phase I and II enzymes. Epidermal growth factor receptor (EGFR) is a cell membrane protein that is overexpressed in many cancers that have a poor prognosis. This cell membrane protein has been considered to be an excellent target for antitumor therapy (27 ). TGF-ß and fibroblast growth factor (FGF) are multifunctional and essential to the survival of cancer cells. They play important roles in promoting cell growth and angiogenesis (28 ,29 ). We observed down-regulation of the EGFR, TGFß2 and FGF by I3C and DIM treatment, demonstrating the inhibitory effects of I3C and DIM on the growth of PC3 prostate cancer cells. Cyclin E, activating transcription factor (ATF) and mitogen-inducible gene (MIG) regulate cell cycle progression, and BCL2 inhibits apoptotic cell death (30 –33 ). The overexpression of ATF has also been observed in metastatic cancer cells (34 ). p57KIP2 is a tumor suppressor that inhibits cyclin-dependent kinase and results in cell cycle arrest (35 ). Our results showed that I3C and DIM inhibited the expression of cyclin E2, ATF5, MIG-2 and BCL2, and induced the expression of p57KIP2, which may lead to the induction of cell cycle arrest and apoptosis. Taken together, I3C and DIM appear to modulate the expression of several genes that may contribute to the observed cell growth inhibition as demonstrated by the MTT cell growth inhibition assay.
Cell signal transduction pathways are important for cell survival. Recently, the MAPK and phosphatidylinositol-3-kinase (PI3K)/Akt pathways have received much attention in cancer research and are believed to be excellent targets for cancer prevention and therapy. The MAPK pathway consists of a three-tiered kinase core in which a MAP3K activates a MAP2K that activates a MAPK, resulting in the activation of NF-B, cell growth and cell survival (36
,37
). We observed down-regulation in the expression of MAP2K3, MAP2K4, MAP4K3, and MARK3 by I3C and DIM treatments, suggesting that I3C and DIM have inhibitory effects on the MAPK pathway, resulting in the abrogation of cancer cell survival. PI3K/Akt pathway is another important signal transduction pathway and plays a critical role in controlling cell survival and apoptosis (38
). From the gene expression profiles of PC3 cells exposed to I3C, we found down-regulation of PI3K expression, suggesting that I3C could induce apoptosis and inhibit cancer cell survival by altering the PI3K/Akt pathway. These findings from cDNA microarray gene analysis are consistent with our previous report showing that I3C inhibits cancer cell growth and induces apoptosis by inhibiting the NF-B and Akt signal pathways (21
,22
).
Several Pol II transcription factors, including transcription factor Dp-1 (TFDP) and NF-YC, play important roles in cell transcription (39 ,40 ). TFDP1 overexpression leads to up-regulation of cyclin E, which encodes a positive regulator for cell cycle G1/S transition (39 ). The overexpression of these transcription factors has also been related to oncogenesis. In addition, core binding factor ß (CBFß) and suppressor of tumorigenicity 16 (ST16) are also involved in oncogenesis. CBFß forms a fusion protein with other gene products and promotes oncogenesis (41 ), whereas ST16 suppresses oncogenesis (42 ). Our results showed that I3C and DIM down-regulated the expression of TFDP1, NF-YC, DKC1, cyclin E and CBFB, and up-regulated ST16 expression, suggesting that I3C and DIM can inhibit transcription and oncogenesis, and could also induce G1 arrest, as demonstrated by our previous study (22 ).
It is important to note, however, that I3C and DIM also had some differential effects on the gene expression profile, which is perhaps expected. We observed that DIM but not I3C induced the expression of other phase II enzymes including methylmalonate-semialdehyde dehydrogenase, phospholipase A2, carbohydrate sulfotransferase 7 and glucuronosyltransferase I, suggesting that DIM may have a more inhibitory effect on oncogenesis than I3C.
In summary, the goal of the present study was to analyze the gene expression profiles of PC3 prostate cancer cells after exposing them to I3C and DIM. Both I3C and DIM changed the expression of a large number of genes that are related to the control of carcinogenesis, cell survival and physiologic behaviors. This may help determine the molecular mechanism(s) by which I3C and DIM exert their pleiotropic effects on PC3 prostate cancer cells; such information could be further exploited for devising chemopreventive and/or therapeutic strategies for prostate cancer.
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FOOTNOTES |
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3 Abbreviations used: ATF, activating transcription factor; CBFß, core binding factor ß; CYP, cytochrome P450; DIM, 3,3'-diindolylmethane; DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; FGF, fibroblast growth factor; I3C, indole-3-carbinol; MAPK, mitogen-activated protein kinase; MAP2K, MAP kinase kinase; MIG, mitogen-inducible gene; NF, nuclear factor; PI3K, phosphatidylinositol-3-kinase; RT-PCR, reverse transcription-polymerase chain reaction; ST16, suppressor of tumorigenicity 16; TFDP, transcription factor Dp-1; TGF-ß, transforming growth factor-ß.
Manuscript received 20 November 2002. Initial review completed 29 December 2002. Revision accepted 21 January 2003.
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