The incidence of late onset breast cancer has been increasing dramatically in the United States. Since genetic factors are believed to result primarily in early onset cases, accumulation of environmental toxins has been proposed as one factor in the increased incidence. Research reported in the Journal of Nutrition demonstrates that EGCG and protect mice against carcinogen induced breast tumors. The green tea polyphenol significantly decreased mammary tumor burden and invasiveness and significantly increased latency to the first tumor. The authors suggest “the ability of EGCG and other tea polyphenols to inhibit carcinogenesis make EGCG a good template for deriving small molecule drugs. Modifications and structure may improve the pharmacokinetics and effectiveness. As a readily available dietary substance, it holds promise for prevention of early-stage cancer.”
Green Tea Polyphenol Epigallocatechin-3 Gallate (EGCG) Affects Gene Expression of Breast Cancer Cells Transformed by the Carcinogen 7,12-Dimethylbenz[a]Anthracene1-3
The Journal of Nutrition
. December 1, 2005 . Sonenshein, Gail E; Taylor, Chad; Yang, Sanghwa; Guo, Shangqin
Since the 1980s, the incidence of late-onset breast cancer has been increasing in the United States. Known risk factors, such as genetic modifications, have been estimated to account for ~5 to 10% of breast cancer cases, and these tend to be early onset. Thus, exposure to and bioaccumulation of ubiquitous environmental chemicals, such as polycyclic aromatic hydrocarbons (PAHs), have been proposed to play a role in this increased incidence. Treatment of female Sprague-Dawley rats with a single dose of the PAH 7,12-dimethylbenz[a]anthracene (DMBA) induces mammary tumors in ~90 to 95% of test animals. We showed previously that female rats treated with DMBA and given green tea as drinking fluid displayed significantly decreased mammary tumor burden and invasiveness and a significantly increased latency to first tumor. Here we used cDNA microarray analysis to elucidate the effects of the green tea polyphenol epigallocatechin-3 gallate (EGCG) on the gene expression profile in a DMBA-transformed breast cancer cell line. RNA was isolated, in quadruplicate, from D3-1 cells treated with 60 Âµg/mL EGCG for 2, 7, or 24 h and subjected to analysis. Semiquantitative RT-PCR and Northern blot analyses confirmed the changes in the expression of 12 representative genes seen in the microarray experiments. Overall, our results documented EGCG-altered expression of genes involved in nuclear and cytoplasmic transport, transformation, redox signaling, response to hypoxia, and PAHs.
J. Nutr. 135: 2978S-2986S, 2005.
KEY WORDS: * EGCG * DMBA * microarray * breast cancer
The rise in breast cancer incidence has been suggested to result in part from increased exposure to and bioaccumulation of lipophilic environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs)5 (1). This hypothesis is based on epidemiological studies relating increased breast cancer to carcinogen exposure (2,3) and from studies showing increased levels of aromatic hydrocarbons and their receptors in breast carcinomas (4,5) and sera from breast cancer patients (2). Furthermore, many studies have shown that PAHs can cause malignant transformation in rodent models in vivo and human mammary cells in vitro. For example, treatment with the PAH 7,12-dimethylbenz[a]anthracene (DMBA) induces mammary tumors in female Sprague-Dawley (S-D) rats (6) and transforms the human mammary epithelial cell line MCF-10F in culture, yielding the D3-1 transformed line (7).
Epidemiological studies indicated that green tea consumption protects against breast cancer (8). Green tea is rich in polyphenols, such as epigallocatechin-3 gallate (EGCG), which possess antioxidant qualities, and were shown to have anticarcinogenic activity against breast and other cancers in animal models. For example, we showed that female S-D rats given green tea as their drinking fluid display a significant decrease in DMBA-induced mammary tumor burden and invasiveness and significantly increased latency to first tumor (9). Similarly, oral consumption of green tea polyphenols was reported to inhibit prostate cancer development and improve survival in the transgenic adenocarcinoma of the mouse prostate TRAMP model (10). To begin to elucidate the exact molecular targets and mechanism for such protection, we turned to breast cancer cell lines as models. We found that EGCG inhibits Her-2/neu receptor tyrosine autophosphorylation in these cancer cells (11). EGCG was also reported to directly inhibit telomerase activity (12,13) and the chymotrypsin-like activity of the proteasome (14). In various models, EGCG was reported to interfere with multiple aspects of control of tumor cell proliferation, apoptosis, angiogenesis, invasion, and metastasis (15-21).
In the present study, we sought to identify the changes in gene expression profile induced by EGCG to probe for the targets mediating the chemopreventive action in DMBA-transformed breast cancer cells using microarray analysis. D3-1 cells were selected because growth of these cells in culture was shown to be potently inhibited by EGCG (9). More recently, EGCG was found to greatly reduce the ability of these cells to grow in soft agar, a hallmark of transformation (data not shown). Our results indicate that genes involved in nuclear and cytoplasmic transport, transformation, redox signaling and hypoxia, and PAH responses were modulated by EGCG.
Materials and methods
Cell culture and mRNA preparation. D3-1 cells were maintained as described previously (9) and grown to 60% confluence for RNA preparation. EGCG (E6234; LKT Laboratory) was dissolved in DMSO. Total RNA was extracted using the UltraspecII RNA isolation kit (Biotex), following the manufacturer’s instructions. The quality of RNA was verified by analyzing RNA samples in a 1% formaldehyde-agarose gel with visualization by ethidium bromide staining.
Reverse transcription and semiquantitative PCR. RNA was digested for 30 min at 37Â°C with RQ1 RNase-Free DNase (Promega), according to the manufacturer’s directions. Briefly, reverse transcription was performed using 5 Âµg total RNA, 1 ÂµL random primers (200 ng), and 1 ÂµL 10 mmol/L deoxyribonucleotide triphosphate (dNTP) mixed in 12 ÂµL, heated to 65Â°C for 5 min, and quick-chilled on ice. All reagents were from InVitrogen unless otherwise specified. Subsequently, 4 ÂµL 5X First-Strand Buffer, 2 ÂµL 0.1 mol/L dithiothreitol, and 1 ÂµL RNasin (Promega) RNAse inhibitor were added. After a 2-min incubation at 42Â°C, 1 ÂµL (200 U) of Superscript reverse transcriptase was added, and the mixture was incubated at 37Â°C for 50 mm. To inactivate the reaction, the samples were heated to 70Â°C for 15 min. Samples (1 ÂµL cDNA) were PCR amplified in a 15 ÂµL reaction volume with 1X reaction buffer (InVitrogen), 2 mmol/L MgCl^sub 2^, 0.2 mmol/L dNTP, 1 Âµmol/L each of primers, and 0.2 ÂµL Taq DNA polymerase. Reactions were performed in a Robocycler PCR machine (Stratagene) or PTC-100 Thermocontroller (MJ Research). The machines were programmed with a 2-min initial denaturing phase at 95Â°C; a cycling phase of 30 s denaturing at 95Â°C, 50 s annealing, and 50 s elongation at 72Â°C; and an extended elongation of 2 min at 72Â°C. Annealing temperature was set at 55Â°C, unless otherwise specified. PCR products from most experiments were resolved on a 1% agarose gel prepared in 40 mmol/L Tris-HCl (pH 8.0), 40 mmol/L acetic acid, and 1 mmol/L EDTA (pH 8.0) containing 0.5 Âµg/mL ethidium bromide or on a 5% polyacrylamide gel using 0.5X TBE running buffer and stained with GelStar nucleic acid stain (Cambrex) for 30 min (22). All gels were visualized with a UV transilluminator and photographed and quantitated with the Kodak DC210 scientific imaging system. The sequences for the primers used for the PCR reactions are listed in Table 1, along with the GenBank accession numbers. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control for RNA integrity and equal loading.
Northern blot analysis. RNA samples (15-20 Âµg) were subjected to Northern blot analysis following published protocols (23). RNA was transferred to GeneScreen Plus (DuPont NEN) by overnight capillary transfer and UV crosslinked. The Xba I restriction fragment DNA from the pcDNAAhR vector (obtained from D. H. Sherr, Boston University School of Medicine), was used as probe for the aryl hydrocarbon receptor (AhR). Ethidium bromide staining of the 18S and 28S rRNA was used to control for equal loading.
cDNA microarray fabrication and hybridisation. A set of 7500 sequence-verified human cDNA clones was purchased from Research Genetics. Bacterial clones were amplified in 96-well culture plates. Plasmid DNA was isolated using a plasmid kit (Millipore) and open reading frames were PCR-amplified using a pair of universal primers, 5′-CTGCAAGGCGATTAAGTTGGGTAAC-3′ and 5′-GTGA-GCGGATAACAATTTC-ACACAGGAAACAGC-3′, under the following conditions: initial denaturation at 94Â°C for 2 min; followed by 30 cycles of 94Â°C for 45 s, 55Â°C for 45 s, and 72Â°C for 2 min; and a final extension step at 72Â°C for 10 min. The PCR amplification products were examined by 1% agarose gel electrophoresis, purified using a Sephadex G-50 column, dried, and then resuspended in a 50% DMSO solution. PCR products were spotted by an OmniGrid(TM) Microarrayer (GeneMachines) onto a silanized glass slide surface (CMT-GAPS; Corning). Each slide was crosslinked with 300 mJ short-wave UV irradiation (Stratalinker) and stored in a desiccator until use.
Target preparation and hybridisation. Control (DMSO) and EGCG-treated D3-1 cells were harvested and total RNA was isolated, as above. RNA from the control DMSO-treated cells was labeled with fluorescent dye cyanine 3-dUTP (Cy3-dUTP; NEN Life Science Products) and used as the control target. RNA from EGCG-treated cells was labeled separately with cyanine 5-dUTP. The labeled cDNAs were purified using a QIAquick PCR Purification Kit (Qiagen) and concentrated through a Microcon-30 column (Millipore) and resuspended in 80 ÂµL hybridization solution (3X SSC and 0.3% SDS). The mixture was denatured at 100Â°C for 2 min and applied to the DNA chip, then incubated at 65Â°C for 16 h in a humidified chamber. The hybridized slide was washed once each in 2X SSC for 2 min; 0.1X SSC, 0.1% SDS for 5 min; and 0.1X SSC for 5 min; then dried by spinning before scanning at room temperature with a GenePix 4000B scanner (Axon Instruments).
Data acquisition, analysis, and statistics. The fluorescence signal was calculated by subtracting the background intensity from the total intensity of a spot using GenePix Pro 4.1 software. Spots with poor signals (F532 – 1.5 Ã B532 < 0 or F635 - 1.5 Ã B635 < 0) were removed from further analysis. Normalization for the expression ratios (median Cy5:median Cy3) was achieved by dividing each ratio by a single normalization factor obtained from the GenePix Pro 4.1 scanning process. Expression ratios for each gene were collected over the time points of each treatment, clustered via the hierarchical clustering method using CLUSTER (24), and visualized using TREEVIEW (24). P-values were calculated assuming that EGCG did not affect gene expression, and the ratio of EGCG/DMSO = 1 using a 2-tailed unequal-variance t-test.
Microarray analysis of EGCG-treated D3-1 cells. DMBA-transformed human D3-1 breast cancer cells were treated with 60 Âµg/mL (130 Âµmol/L) of EGCG or an equivalent amount of carrier solution DMSO for 2, 7, or 24 h, and total RNA was isolated and subjected to microarray analysis using a cDNA array with 7500 human genes. The experiment was performed in duplicate twice, resulting in quadruplicate replication of each time point. A heat map of one experiment is presented in Figure 1. The same genes that appeared to change in all experiments are summarized in Figure 2. Columns 1 to 4 designate each of the quadruplicate readings in the corresponding experiment. The numbers in the shaded area show the gene expression changes in samples treated with EGCG versus DMSO. Values > 1, shaded in dark grey, indicate genes that were upregulated by EGCG treatment, compared with the control DMSO treatment. Values < 1, shaded in light grey, indicate genes that were downregulated by EGCG treatment, versus the control treatment. Values = 1 indicate that the treatment and control samples did not differ. The gene ontology information was obtained from the Genetics Department at Stanford University (25), and gene information for Homo sapiens was obtained from the National Library of Medicine (26).
Genes that increased or decreased separated very well by 7 h; at 2 h there was considerable variability between the quadruplicate samples, perhaps because of an initial early change that was reversed later. Overall, the direction of change was maintained to 24 h of treatment. Gene changes at 24 h were chosen for further study. Several housekeeping genes were analyzed similarly to controls [including ring finger protein 5 (AA402960), inosine monophosphate dehydrogenase 2 (N73268), soluble acid phosphatase 1 (W45148), and cyclophillin (AA418410)]; these showed no variation with EGCG, as expected (data not shown). Overall, significant changes were detected in 21 genes.
LIM and SH3 protein 1 (AI003699), hypoxia upregulated 1 (AA099134), AhR (AA181307), rab3 GTPase-activating protein (AA520985), myeloid cell leukemia sequence 1 (Mcl1; AA488674), tight junction protein 1 (H50344), thrombospondin 1 (AA464532), sterol response element binding protein 2 (SREBP2; AA053886), metallothionein 1E (AA872383), human clone 23721 mRNA sequence (R45056), Ras-GTPase activating protein SH3 domain-binding protein 2 (AA151214), chromosome segregation 1-like (CSE-1; N69204), karyopherin a 6 (AI865149), LanC-like 1 (R59621), nucleosome assembly protein 1-like 4 (H92201), chord domain-containing protein 1 (AA773461), and solute family carrier protein 20 member 1 (W46972) were all downregulated by EGCG at 24 h. In contrast, aldo-keto reductase (AKR) family 1, C3 (AA916325), AKR family 1, C2 (AI924357), AKR family 1, C1 (R93124), carbonic anhydrase IX (AI023541) and peroxisome proliferator activated receptor (PPAR)γ angiopoietin-related protein (T54298) were all upregulated by EGCG at 24 h. Although changes in other genes were seen, they did not appear to reach statistical significance; these included protein tyrosine phosphatase, nonreceptor type 11 (PTPN11; AA995560), epithelial cell transforming sequence (ECTS) 2 oncogene (AI031571), H1 histone family, member 0 (W69399), and connective tissue growth factor (CTGF; AA598794).
Confirmation of gene expression changes induced by EGCG. RT-PCR analyses were performed to validate the changes in gene expression seen in the microarray analysis. RNA was freshly isolated from D3-1 cells treated with 60 Âµg/mL EGCG for 24 h and processed for RT-PCR using primer sequences specified in Table 1. A panel of 13 genes was selected for confirmation, in addition to ring finger protein 5 and GAPDH as controls (Fig. 3). Seven of the genes changed in both replicate experiments. The expression of the other 6 genes changed in only 2 of the 4 experiments, and these were selected for their potential relevance to breast cancer. As shown in Figure 3, RT-PCR confirmed most of the changes in gene expression observed with the microarray analysis. In particular, CSE-1, CTGF, AhR, LIM and SH3 protein 1, hypoxia upregulated 1, rab3 GTPase-activating protein, myeloid cell leukemia sequence, tight junction protein 1, SREBP2, PTPN11, metallothionein 1E, epithelial cell transforming sequence 2 oncogene, thrombospondin 1, human clone 23721 mRNA sequence, Ras-GTPase activating protein SH3 domain-binding protein 2, H1 histone family member 0, karyopherin α 6, LanC-like 1, nucleosome assembly protein 1-like 4, and chord domain-containing protein 1 were all downregulated by EGCG at 24 h. In contrast, AKR family 1 C3, C2, and C1; carbonic anhydrase IX; and PPARγ angiopoietin-related protein were all upregulated by EGCG at 24 h. The housekeeping gene ring finger protein 5 (AA402960), which did not change in the microarray, showed no change in expression by RT-PCR assay (Fig. 3). Analysis of GAPDH, which was included as an additional control, confirmed equal RNA loading. Lastly, Northern blot analysis, which was performed to assess AhR mRNA levels (Fig. 4), confirmed significant downregulation of AhR gene expression upon EGCG treatment.
Some genes seen to change in only 1 of the 2 replicate experiments were tested by RT-PCR for confirmation. These included bone morphogenic protein 6 (BMP6; AA424833), glutathione S-transferase (GST) A4 (AA152347), transforming growth factor-β1 (TGF-β1; R36467), and Wnt signaling inducible secreted protein 1 (WISP-1; (AI473336), which were all upregulated, and heat shock protein 10 kDa (HSP10; AA448396) and PTPN11 (AA995560), which were downregulated by EGCG treatment at 24 h. RNA expression of BMP6, GST A4, TGF-β1, and WISP-1 were all shown to increase by RT-PCR, whereas HSP10 was shown to decrease. In contrast, PTPN11 did not show any substantial change when assayed by RT-PCR, consistent with the original statistical analysis. Thus, overall, the RNA analysis largely confirmed the changes in gene expression identified by the microarray analysis.
In the present study, we demonstrated that EGCG treatment of D3-1 breast cancer cells mediated changes in gene expression that promote a more normal phenotype. In particular, microarray analyses demonstrated that genes involved in nuclear and cytoplasmic transport, transformation, redox signaling, and hypoxia and PAH signaling responses were modulated by EGCG, indicating an overall chemopreventive role of EGCG, although a few minor exceptions were noted. Below, we discuss the potential physiological significance of these changes.
Downregulated genes. Two of the genes downregulated by EGCG encode proteins involved in nucleocytoplasmic transport: CSE-1 and karyopherin α 6 (Table 2). The nuclear localization signal (NLS) functions via interaction with the NLS import receptor, a heterodimer of importin α and β subunits, also known as karyopherins. Importin α binds the NLS-containing cargo in the cytoplasm, and importin β docks the complex at the cytoplasmic side of the nuclear pore complex. In the presence of nucleoside triphosphates and the small GTP-binding protein Ran, the complex moves into the nuclear pore complex, and the importin subunits dissociate. Importin α enters the nucleoplasm with its passenger protein, and importin β remains at the pore. CSE-1 is an export receptor for importin a, mediating importin a reexport from the nucleus to the cytoplasm after it has released its load into the nucleoplasm (27). CSE-1 was isolated as cDNA fragments that render MCF-7 breast cancer cells resistant to cell death caused by pseudomonas exotoxin, diphtheria toxin, and tumor necrosis factor (28). Its expression is low in quiescence or on growth arrest and is highly expressed in actively dividing cells, including tumor cell lines (28), consistent with its reduced expression in the presence of EGCG. Karyopherin a 6 (importin α 7) encodes a member of the importin a family. The decreases in CSE-1 and karyopherin α 6 gene expression were significant, and the change in CSE-1 was confirmed by RT-PCR analysis.
A decrease in AhR expression was confirmed by RT-PCR and Northern blot analysis. The AhR is a cytosolic, ligand-activated receptor and transcription factor involved in the regulation of biological responses to several classes of carcinogenic environmental chemicals (e.g., DMBA and other PAHs, dioxin, and planar polychlorinated biphenyls). On activation, the receptor moves to the nucleus in a complex and induces gene transcription mediated by xenobiotic response elements, including those encoding the cytochrome P450 (CYP) enzymes CYP1A1, CYP1A2, and CYP1B1. High levels of constitutively active AhR were found in human breast cancer specimens and in DMBA-induced rat mammary tumors, and its induction occurred early in the DMBA-induced carcinogenesis (4). If EGCG similarly decreases AhR levels in the mammary glands of S-D rats, this could contribute to the observed decrease in tumor burden resulting from DMBA treatment in the rats given green tea as their drinking fluid (9). Work from our laboratory has shown a functional interaction between AhR and classical nuclear factor-κB (NF-κB), which cooperatively transactivate the c-myc oncogene (29). The reduction in the expression of AhR thus might compromise the NF-κB activity observed in these cells (29), decreasing its full oncogenic potential. Interestingly, the downregulation of AhR by EGCG was not seen in the Her-2/neu-overexpressing NF639 cells (data not shown), suggesting that the primary targets of GTPs are different depending on cell types, are related to the etiology of transformation, or both.
Although the decrease in mRNA levels of the 2 growth-promoting factors, CTGF and ECTS, was not significant when measured by microarray analysis, a clear reduction was measured by RT-PCR. CTGF is the major connective tissue mitoattractant secreted by vascular endothelial cells. Advanced breast cancers were found to overexpress CTGF by Xie et al. (30) whereas Jiang et al. (31) detected a reduced level. CTGF is induced by hypoxia, and recent evidence implicates HIF1α in direct regulation of CTGF promoter activity (32). Interestingly, hypoxia upregulated 1 gene product, a member of the heat shock protein 70 (HSP70) family, has an important cytoprotective role in hypoxia-induced cellular perturbations (33). The hypoxia upregulated 1 gene product plays an important role in protein folding and secretion in the endoplasmic reticulum, is upregulated in breast tumors, and is associated with tumor invasiveness. Expression of this gene was also significantly reduced by EGCG in the microarray analysis (~3-fold, P = 0.003). ECTS is a transforming protein related to Rho-specific exchange factors and yeast cell cycle regulators. It is expressed in a cell cycle-dependent manner during liver regeneration and plays an important role in the regulation of cytokinesis (34,35).
Expression of several additional genes displayed significant decreases in the microarray analyses as yet not confirmed by RT-PCR. For example, SREBP2 was downregulated ~2-fold by EGCG (P = 0.003). SREBPs are master transcription regulators for many important genes involved in metabolism. SREBP expression increases during malignant transformation, leading to increased expression of genes involved in lipid metabolism to sustain accelerated tumor cell growth (36). Fatty acid synthase is overexpressed in several human cancers, and inhibition of fatty acid synthase suppresses Her-2/neu overexpression in cancer cells (37). SREBP and its downstream effecter genes are upregulated during progression to androgen independence in prostate cancer models (38). Mcl-1, which is in the Bcl-2 family, is involved in the programming of differentiation and concomitant maintenance of viability. In breast cancer cells and myeloma cells, Mcl-1 possesses strong antiapoptotic function (39,40). Thus, inhibition of antiapoptotic signals might be another mechanism for EGCG to inhibit tumor formation. LIM and SH3 protein 1 mRNA encodes a member of a LIM protein subfamily, which is characterized by a LIM motif and a SH3 domain. It is overexpressed in breast cancers (41). Thrombospondin 1, HSP10, tight junction protein 1, H1 histone family member 0, nucleosome assembly protein 1-like 4, and prefoldin are other gene products that were downregulated by EGCG. The primary known cellular functions of these genes are briefly discussed in Table 2. Metallothionein 1E, LanC-like 1 (bacterial), Ras-GTPase activating protein SH3 domain-binding protein 2, Rab3 GTPase-activating protein, cysteine and histidine-rich domain-containing protein 1, prefoldin, and solute carrier family 20 (phosphate transporter) member 1 are other genes that appeared downregulated by EGCG. Although the functions and regulation of these proteins are less well understood, their collective modulation by EGCG may represent pathways for EGCG to exert its anticarcinogenic function in DMBA-induced transformation.
Upregulated genes. A brief summary of the primary functions of genes that were upregulated by EGCG is given in Table 3. EGCG induced expression of 3 of the 4 isoforms of 3α-hydroxysteroid dehydrogenases or AKRs across the time course: AKR C1, AKR C2, and AKR C3. These enzymes catalyze the conversion of aldehydes and ketones to their corresponding alcohols, using NADH, NADPH, or both as cofactors (42). They inactivate steroid hormones in the liver, regulate 5α-dihydrotestosterone levels in the prostate, and form the neurosteroid allopregnanolone in the central nervous system. These enzymes have also been implicated in the metabolic activation of PAH trans-dihydrodiols, which cause cytotoxicity. Overexpression of this class of enzyme in MCF-7 cells led to cell death (43). AKR1C4 oxidized DMBA-3,4-diol to the highly reactive DMBA-3,4-dione (44). The collective upregulation in the AKRs by EGCG may be reflective of the D3-1 cell etiology, because these cells were transformed by DMBA in vitro. The changes identified by microarray in all 3 AKRs were significant (AKR C3, P = 0.007; AKR C2, P = 0.008; and AKR C1, P = 0.001).
The increase in PPARγ angiopoietin-related protein and carbonic anhydrase (CA)IX genes may reflect changes in hypoxia-induced pathways. PPARγ angiopoietin-related protein shows hypoxia-induced expression in endothelial cells and plays important roles in angiogenesis (45). CAIX, which is membrane associated, is strongly induced by hypoxia. CAIX is overexpressed in a variety of tumor types and associated with increased metastasis and poor prognosis (46). The regulation of most proteins required for hypoxic adaptation occurs at the gene level, which involves transcriptional induction via the binding of a transcription factor HIF-1 to the hypoxia-response element on the regulated genes (47). However, the upregulation of HIF-1 itself was not seen in the microarray analysis. Additional analysis will be required to elucidate the mechanism of CAIX mRNA induction.
At present, 8 distinct classes of the soluble cytoplasmic mammalian GSTs have been identified: α, κ, μ, Ω, τ, σ, θ, and ζ. These enzymes are involved in cellular defense against toxic, carcinogenic, and pharmacologically active electrophilic compounds. GST A4 encodes a member belonging to the a class. It is distinguished by high catalytic efficiency toward the substrate 4-hydroxynon-2-enal, a cytotoxic and mutagenic lipid peroxidation product of oxidative stress (48). The upregulation of GST A4 induced by EGCG might be related to balancing the cellular redox status perturbed by EGCG, which is known to possess strong antioxidative capacity (49). Interestingly, members of the μ and θ classes GST Âµl (GSTM1) and GST θ1 (GSTT1) have been implicated in the sensitivity to green tea as an agent to prevent oxidative damage (50).
The increase in TGF-β1 mRNA is particularly interesting. TGF-β1 is synthesized as a precursor, which requires processing to control proliferation, and epithelial-to-mesenchymal transition (EMT). Although TGF-β1 inhibits NF-κB activity and slows growth or induces apoptosis in less transformed cells (51,52), it promotes EMT of Ras-transformed cells (53). Interestingly, we showed that NF-κB activity in Ras-transformed liver epithelial cells is resistant to inhibition by TGF-β1 (54). The primary known cellular functions of the above-discussed proteins, as well as those of WISP-1 and BMP6, are briefly given in Table 3.
Taken together, EGCG treatment induced changes in expression of a large number of genes that have potential relevance to tumor biology. Other groups have used microarray analysis to study the action of EGCG in other cellular systems. Human lung cancer cell line PC-9 cells were treated with 200 Âµmol/L (92 Âµg/mL) of EGCG for 7 h, and gene expression was profiled using an Atlas Human Cancer cDNA Expression Array containing 588 genes (55). Human prostate cancer cell line LNCaP cells treated with 12 Âµmol/L (5.5 Âµg/mL) EGCG for 12 h were analyzed using a Micromax Direct System (56,57). In another study, human papillomavirus-16-associated cervical cancer cell line CaSki cells were treated with 35 Âµmol/L (16 Âµg/mL) of EGCG for 12, 24, and 48 h, and gene expression was profiled using a Macrogen 384-cDNA chip (16). Human vascular endothelial cells were exposed to green tea extracts for 6 and 48 h, and gene expression was profiled using an Affymetrix chip containing 12,625 genes (58). These studies reported genes up- or downregulated by >2-fold. These included gene categories involved in proliferation control, cell cycle control, and apoptosis, confirming the findings with conventional molecular and cellular biology studies. However, the differences between the cell types, the duration and dose of treatment, and the array chips used for these experiments make a direct comparison almost impossible. Our current data provide a catalogue of genes involved in breast cancer, with particular emphasis on the DMBA-transformed etiology.
In addition to EGCG, the effects of other green tea polyphenols including epicatechin, epicatechin-3-gallate, and epigallocatechin are also of interest for examination, because they have been reported to have anticarcinogenic activity as well. The ability of EGCG and other tea polyphenols to inhibit carcinogenesis make EGCG a good template for deriving small-molecule drugs. Modifications in structure may improve the pharmacokinetics and effectiveness. As a readily available dietary substance, it holds promise for prevention of early-stage cancer. Our very recent studies with the DMBA-induced mammary tumorigenesis model demonstrated that in situ tumors in rats drinking green tea versus water have a less-invasive phenotype (unpublished observation). New target identification with gene expression profiling may help in designing new effective adjuvant therapy treatments. The present study was designed to evaluate the protective effect of EGCG on a specific environmental carcinogen (DMBA). It would be important to also evaluate the protective effect in other oncogenic settings. For example, we previously showed that EGCG inhibits Her-2/neu receptor tyrosine phosphorylation and downstream signaling. These findings suggest that EGCG may also be effective in the treatment of breast cancer overexpressing this oncogene, especially when combined with other chemotherapeutic agents. It would be of great interest to compare the data from the present study with similar high-throughput analysis of Her-2/neu tumors with regard to the different and common target genes for EGCG. These studies would allow for identification of key molecules and pathways and provide a list of candidate genes whose functional role might be critical for the chemopreventive and antiinvasive role of green tea polyphenols.
We thank Zidong Zhang for technical assistance in performing the RT-PCR analysis, and D. H. Sherr, Boston University School of Medicine, for generously providing cloned AhR DNA.
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Shangqin Quo, Sanghwa Yang,* Chad Taylor, and Gail E. Sonenshein4
Department of Biochemistry and Women’s Health Interdisciplinary Research Center, Boston University School of Medicine, Boston, MA 02118-2394; and * Cancer Metastasis Research Center, Yonsei University College of Medicine, Seodaemun-Gu, Seoul 120-752, Korea
4 To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.