| Taste the difference! |
Green Tea Polyphenol Epigallocatechin-3 Gallate (EGCG) Affects Gene
Expression of Breast Cancer Cells Transformed By the Carcinogen 7,12-
Dimethylbenz[a]Anthracene1-3
By Guo, Shangqin; Yang, Sanghwa; Taylor, Chad; Sonenshein, Gail E
ABSTRACT
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 g/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 37C
with RQ1 RNase-Free DNase (Promega), according to the manufacturer's directions.
Briefly, reverse transcription was performed using 5 g total RNA, 1 L random primers
(200 ng), and 1 L 10 mmol/L deoxyribonucleotide triphosphate (dNTP) mixed in 12 L,
heated to 65C for 5 min, and quick-chilled on ice. All reagents were from InVitrogen
unless otherwise specified. Subsequently, 4 L 5X First-Strand Buffer, 2 L 0.1 mol/L
dithiothreitol, and 1 L RNasin (Promega) RNAse inhibitor were added. After a 2-min
incubation at 42C, 1 L (200 U) of Superscript reverse transcriptase was added, and the
mixture was incubated at 37C for 50 mm. To inactivate the reaction, the samples were
heated to 70C for 15 min. Samples (1 L cDNA) were PCR amplified in a 15 L reaction
volume with 1X reaction buffer (InVitrogen), 2 mmol/L MgCl^sub 2^, 0.2 mmol/L dNTP, 1
mol/L each of primers, and 0.2 L 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 95C; a cycling
phase of 30 s denaturing at 95C, 50 s annealing, and 50 s elongation at 72C; and an
extended elongation of 2 min at 72C. Annealing temperature was set at 55C, 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 g/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.
TABLE 1
Primer sequences used in RT-PCR analyses
Northern blot analysis. RNA samples (15-20 g) 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 94C for
2 min; followed by 30 cycles of 94C for 45 s, 55C for 45 s, and 72C for 2 min; and a
final extension step at 72C 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 L hybridization solution (3X SSC and 0.3%
SDS). The mixture was denatured at100C for 2 min and applied to the DNA chip, then
incubated at 65C 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 B532 < 0 or F635 - 1.5 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.
Results
Microarray analysis of EGCG-treated D3-1 cells. DMBA-transformed human D3-1
breast cancer cells were treated with 60 g/mL (130 mol/ 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).
FIGURE 1 Heat-map data from a representative microarray analysis. D3-1 cells were
treated with 60 g/mL EGCG for 2, 7 or 24 h, and samples of total RNA were subjected
to microarray analysis using a cDNA array with 7500 human genes (GenomicTree).
Colors indicate that expression was upregulated (red) or downregulated (green) by
EGCG treatment, compared with control DMSO treatment; that samples did not differ
between EGCG and control treatment (black); or that there was no detectable signal
(gray).
FIGURE 2 Summary of genes changed in all experiments. Data were generated from
two independent experiments with D3-1 cells treated, in duplicate, with EGCG or
DMSO, as described in Figure 1. Replicate analyses 1 to 4 for each time point and the
quadruplicate samples for each time point are grouped together. Numbers in the
shaded areas indicate fold change in cells treated with EGCG. compared with control
DMSO treatment. Values > 1 indicate genes upregulated by EGCG (dark grey); values
< 1 indicate downregulation by EGCG (light grey). Values = 1 indicate that EGCG
treatment did not differ from control (no shading). P-values are calculated for the 24-h
time point; 2-tailed unequal-variance t-test, assuming EGCG causes a 1- fold change
in gene expression.
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 g/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.
FIGURE 3 RT-PCR assessment of changes in RNA levels. D3-1 cells were treated with
60 g/mL EGCG for 24 h, and total RNA samples (5 g) were processed for reverse
transcription. The primer sequences for each gene are given in Table 1. PCR
conditions were determined by pilot experiments for the quantitative phase. The PCR
products were either resolved on an agarose gel and visualized with ethidium bromide
or resolved on a polyacrylamide gel and visualized by GelStar. *Genes presented in
Figure 2.
FIGURE 4 Northern blot analysis of AhR mRNA levels. D3-1 cells were treated with 60
g/mL of EGCG for 24 h and total RNA was harvested. Samples (20 g RNA) were
resolved in a 1% formamide- agarose gel and visualized by ethidium bromide staining.
As controls for integrity and equal loading, 28S and 18S RNAs were recorded using the
Kodak Digital Camera DC210 system. The gel was then processed for Northern blot
analysis using as probe a 3.2-kb AhR cDNA fragment obtained by Xba I digestion of
the pcDNAAhR vector.
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.
Discussion
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 exceptionswere 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).
TABLE 2
Summary of genes in D3-1 cells whose expression is decreased by 24-h treatment with
EGCG
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).
TABLE 3
Summary of genes in D3-1 cells whose expression is increased by 24-hour treatment
with EGCG
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 l (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 induc\es 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 mol/L (92 g/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 mol/L (5.5 g/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 mol/L (16 g/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.
ACKNOWLEDGMENTS
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.
1 Published in a supplement to The Journal of Nutrition. Presented as part of the
International Research Conference on Food, Nutrition, and Cancer held in
Washington, DC, July 14-15, 2005. This conference was organized by the American
Institute for Cancer Research and the World Cancer Research Fund International and
sponsored by (in alphabetical order) California Avocado Commission; California Walnut
Commission; Campbell Soup Company; The Cranberry Institute; Danisco USA, Inc.;
The Hormel Institute; National Fisheries Institute; The Solae Company; and United
Soybean Board. Guest editors for this symposium were Vay Liang W. Go, Ritva R.
Butrum, and Helen A. Norman. Guest Editor Disclosure: R. R. Butrum and H. Norman
are employed by conference sponsor American Institute for Cancer Research; V.L.W.
Go, no relationships to disclose.
2 Author Disclosure: No relationships to disclose.
3 Supported by NIH, Grant PO1 ES11624 (GES), and the Korean Ministry of Science
and Technology Basic Research Program (Korean Science and Engineering
Foundation), Grant 2004-2-0847 (SY).
5 Abbreviations used: AhR, aryl hydrocarbon receptor; AKR, aldo- keto reductase;
BMP6, bone morphogenic protein 6; CA, carbonic anhydrase; CSE-1, chromosome
segregation 1-like; CTGF, connective tissue growth factor; CYP, cytochrome P450;
DMBA, 7,12- dimethylbenz[a]anthracene; dNTP, deoxyribonucleotide triphosphate;
EGCG, epigallocatechin-3 gallate; EMT, epithelial-to-mesenchymal transition; ECTS,
epithelial cell transforming sequence; GAPDH, glyceraldehyde 3-phosphate
dehydrogenase; GST, glutathione S- transferase; HSP10, heat shock protein 10 kDa;
HSP70, heat shock protein 70 kDa; Mcl1, myeloid cell leukemia sequence 1; NF-κB,
nuclear factor-κB; NLS, nuclear localization signal; PAH, polycyclic aromatic
hydrocarbon; PPAR, peroxisome proliferator activated receptor; PTPN11, protein
tyrosine phosphatase nonreceptor type 11; S-D, Sprague-Dawley; SREBP2, sterol
response element binding protein 2; TGF-β1, transforming growth factor-β1; WISP-1,
Wnt signaling inducible secreted protein 1.
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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: gsonensh@bu.edu.
Copyright American Institute of Nutrition Dec 2005
Expression of Breast Cancer Cells Transformed By the Carcinogen 7,12-
Dimethylbenz[a]Anthracene1-3
By Guo, Shangqin; Yang, Sanghwa; Taylor, Chad; Sonenshein, Gail E
ABSTRACT
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 g/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 37C
with RQ1 RNase-Free DNase (Promega), according to the manufacturer's directions.
Briefly, reverse transcription was performed using 5 g total RNA, 1 L random primers
(200 ng), and 1 L 10 mmol/L deoxyribonucleotide triphosphate (dNTP) mixed in 12 L,
heated to 65C for 5 min, and quick-chilled on ice. All reagents were from InVitrogen
unless otherwise specified. Subsequently, 4 L 5X First-Strand Buffer, 2 L 0.1 mol/L
dithiothreitol, and 1 L RNasin (Promega) RNAse inhibitor were added. After a 2-min
incubation at 42C, 1 L (200 U) of Superscript reverse transcriptase was added, and the
mixture was incubated at 37C for 50 mm. To inactivate the reaction, the samples were
heated to 70C for 15 min. Samples (1 L cDNA) were PCR amplified in a 15 L reaction
volume with 1X reaction buffer (InVitrogen), 2 mmol/L MgCl^sub 2^, 0.2 mmol/L dNTP, 1
mol/L each of primers, and 0.2 L 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 95C; a cycling
phase of 30 s denaturing at 95C, 50 s annealing, and 50 s elongation at 72C; and an
extended elongation of 2 min at 72C. Annealing temperature was set at 55C, 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 g/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.
TABLE 1
Primer sequences used in RT-PCR analyses
Northern blot analysis. RNA samples (15-20 g) 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 94C for
2 min; followed by 30 cycles of 94C for 45 s, 55C for 45 s, and 72C for 2 min; and a
final extension step at 72C 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 L hybridization solution (3X SSC and 0.3%
SDS). The mixture was denatured at100C for 2 min and applied to the DNA chip, then
incubated at 65C 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 B532 < 0 or F635 - 1.5 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.
Results
Microarray analysis of EGCG-treated D3-1 cells. DMBA-transformed human D3-1
breast cancer cells were treated with 60 g/mL (130 mol/ 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).
FIGURE 1 Heat-map data from a representative microarray analysis. D3-1 cells were
treated with 60 g/mL EGCG for 2, 7 or 24 h, and samples of total RNA were subjected
to microarray analysis using a cDNA array with 7500 human genes (GenomicTree).
Colors indicate that expression was upregulated (red) or downregulated (green) by
EGCG treatment, compared with control DMSO treatment; that samples did not differ
between EGCG and control treatment (black); or that there was no detectable signal
(gray).
FIGURE 2 Summary of genes changed in all experiments. Data were generated from
two independent experiments with D3-1 cells treated, in duplicate, with EGCG or
DMSO, as described in Figure 1. Replicate analyses 1 to 4 for each time point and the
quadruplicate samples for each time point are grouped together. Numbers in the
shaded areas indicate fold change in cells treated with EGCG. compared with control
DMSO treatment. Values > 1 indicate genes upregulated by EGCG (dark grey); values
< 1 indicate downregulation by EGCG (light grey). Values = 1 indicate that EGCG
treatment did not differ from control (no shading). P-values are calculated for the 24-h
time point; 2-tailed unequal-variance t-test, assuming EGCG causes a 1- fold change
in gene expression.
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 g/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.
FIGURE 3 RT-PCR assessment of changes in RNA levels. D3-1 cells were treated with
60 g/mL EGCG for 24 h, and total RNA samples (5 g) were processed for reverse
transcription. The primer sequences for each gene are given in Table 1. PCR
conditions were determined by pilot experiments for the quantitative phase. The PCR
products were either resolved on an agarose gel and visualized with ethidium bromide
or resolved on a polyacrylamide gel and visualized by GelStar. *Genes presented in
Figure 2.
FIGURE 4 Northern blot analysis of AhR mRNA levels. D3-1 cells were treated with 60
g/mL of EGCG for 24 h and total RNA was harvested. Samples (20 g RNA) were
resolved in a 1% formamide- agarose gel and visualized by ethidium bromide staining.
As controls for integrity and equal loading, 28S and 18S RNAs were recorded using the
Kodak Digital Camera DC210 system. The gel was then processed for Northern blot
analysis using as probe a 3.2-kb AhR cDNA fragment obtained by Xba I digestion of
the pcDNAAhR vector.
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.
Discussion
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 exceptionswere 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).
TABLE 2
Summary of genes in D3-1 cells whose expression is decreased by 24-h treatment with
EGCG
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).
TABLE 3
Summary of genes in D3-1 cells whose expression is increased by 24-hour treatment
with EGCG
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 l (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 induc\es 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 mol/L (92 g/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 mol/L (5.5 g/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 mol/L (16 g/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.
ACKNOWLEDGMENTS
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.
1 Published in a supplement to The Journal of Nutrition. Presented as part of the
International Research Conference on Food, Nutrition, and Cancer held in
Washington, DC, July 14-15, 2005. This conference was organized by the American
Institute for Cancer Research and the World Cancer Research Fund International and
sponsored by (in alphabetical order) California Avocado Commission; California Walnut
Commission; Campbell Soup Company; The Cranberry Institute; Danisco USA, Inc.;
The Hormel Institute; National Fisheries Institute; The Solae Company; and United
Soybean Board. Guest editors for this symposium were Vay Liang W. Go, Ritva R.
Butrum, and Helen A. Norman. Guest Editor Disclosure: R. R. Butrum and H. Norman
are employed by conference sponsor American Institute for Cancer Research; V.L.W.
Go, no relationships to disclose.
2 Author Disclosure: No relationships to disclose.
3 Supported by NIH, Grant PO1 ES11624 (GES), and the Korean Ministry of Science
and Technology Basic Research Program (Korean Science and Engineering
Foundation), Grant 2004-2-0847 (SY).
5 Abbreviations used: AhR, aryl hydrocarbon receptor; AKR, aldo- keto reductase;
BMP6, bone morphogenic protein 6; CA, carbonic anhydrase; CSE-1, chromosome
segregation 1-like; CTGF, connective tissue growth factor; CYP, cytochrome P450;
DMBA, 7,12- dimethylbenz[a]anthracene; dNTP, deoxyribonucleotide triphosphate;
EGCG, epigallocatechin-3 gallate; EMT, epithelial-to-mesenchymal transition; ECTS,
epithelial cell transforming sequence; GAPDH, glyceraldehyde 3-phosphate
dehydrogenase; GST, glutathione S- transferase; HSP10, heat shock protein 10 kDa;
HSP70, heat shock protein 70 kDa; Mcl1, myeloid cell leukemia sequence 1; NF-κB,
nuclear factor-κB; NLS, nuclear localization signal; PAH, polycyclic aromatic
hydrocarbon; PPAR, peroxisome proliferator activated receptor; PTPN11, protein
tyrosine phosphatase nonreceptor type 11; S-D, Sprague-Dawley; SREBP2, sterol
response element binding protein 2; TGF-β1, transforming growth factor-β1; WISP-1,
Wnt signaling inducible secreted protein 1.
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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: gsonensh@bu.edu.
Copyright American Institute of Nutrition Dec 2005
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