Background
Numerous experimental and clinical studies have established that the clinical outcome of chemotherapeutic strategies for breast cancer commonly rely on the expression of important growth factor receptors such as the nuclear estrogen receptors (ERs) [
1]. ERs mediate effects of estrogen hormone such as 17β-estradiol (E
2) through a ligand-receptor binding activated signal pathway leading to cellular proliferation and differentiation in normal mammary tissue [
2]. The status of ERs also plays an important role in monitoring of the malignant behavior of breast cancer. Of the two major isoforms of ERs (ERα and ERβ) that have been identified to date, however, the ERα isoform is believed to primarily contribute to estrogen induced growth-stimulatory effects in breast cancer [
3]. For the tumors that express ERα, therapeutic strategies include estrogen ablation or anti-estrogens. However, ERα-negative breast cancers have more clinically aggressive biological characteristics and the prognosis is poor because of the lack of target-directed therapies [
4].
It has been known that the absence of ERα gene expression in ERα-negative breast cancer is not due to DNA mutations of the ERα gene [
5]. Therefore, acquired loss of ERα transcription is a potential mechanism for hormone resistance in ERα-negative breast cancer. Previous studies have shown that more than 25% of ERα-negative breast cancer cells have an aberrant methylation status of the ERα promoter [
6‐
8]. In addition, histone acetylation/deacetylation has also been implicated as a common mechanism underlying ERα gene trans-activation/repression in human malignant mammary cells [
9]. Correspondingly, DNA methyltranferase (DNMT) inhibitors such as 5-aza-2'-deoxycytidine (5-aza) and histone deacetylase (HDAC) inhibitors like trichostatin A (TSA) have been successfully used to induce ER expression and sensitize hormone-resistant ERα-negative breast cancer cells to chemotherapy [
8,
10‐
12]. In this regard, it is increasingly evident that epigenetic events play an important role in ERα gene expression.
Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, has been extensively studied as a bioactive dietary component against various types of carcinomas through multiple mechanisms such as anti-oxidation, induction of apoptosis, inhibition of angiogenesis and metastasis [
13]. It has been shown that EGCG can prevent and inhibit breast tumorigenesis independently of ER status [
14,
15]. Moreover, EGCG enhanced tamoxifen-induced cellular apoptosis in ERα-negative MDA-MB-231 breast cancer cells suggesting that EGCG may impart its anti-cancer property through a unique mechanism acting on ERα signal transduction [
16]. However, the precise molecular mechanisms underlying this phenomenon are still unclear. Recently, one potential mechanism that has received considerable attention is that EGCG can modulate gene expression by influencing epigenetic processes such as DNA methylation and/or histone modification [
17,
18]. Studies have shown that EGCG can alter DNA methylation patterns in human cancer cells as well as mouse models and by directly and indirectly inhibiting the enzymatic activities of DNA methyltransferases (DNMTs) [
18,
19]. This effect results in reactivation of methylated-silencing tumor suppressor genes such as
p16
INK4a
, retinoic acid receptor β (
RAR β), and the DNA mismatch repair gene human
mutL homologue 1 (
hMLH1) which collectively leads to tumor suppression [
18]. Furthermore, it is believed that EGCG-induced remodeling of chromatin structure is a key epigenetic mechanism for regulating tumor-related gene transcription. Consistently, our previous studies also found that the green tea polyphenol, EGCG, can influence patterns of histone acetylation in the human telomerase reverse transcriptase (
hTERT) promoter, which leads to
hTERT transcription inhibition and tumor suppression in malignant human mammary cells [
17]. Since estrogen-resistant breast cancers pose a major risk to breast cancer patients, we asked whether EGCG may facilitate the epigenetic processes leading to ERα re-expression in ERα-negative breast cancer cells and whether combination epigenetic approaches may have synergistic effects in these cells.
Our studies were aimed to address the epigenetic mechanisms of ERα reactivation by EGCG in hormone-resistant breast cancer cells. In the present studies, we analyzed the epigenetic mechanisms of ERα re-expression and corresponding ERα-stimulated signal pathway in ERα-negative MDA-MB-231 cells treated with EGCG. In addition, by applying two epigenetic modulators including the HDAC inhibitor, TSA and the demethylation agent, 5-aza, we were able to investigate the epigenetic mechanisms of ERα-reactivation and to explore the applicability of this or similar combination to breast cancer therapy. We found, for the first time, that EGCG and TSA can synergistically reactivate ERα expression and thus, activate the ERα binding-induced cellular signal pathway through epigenetic control. Clinically, this reactivation of ERα enhances chemosensitivity to tamoxifen, an anti-estrogen drug, in ERα-negative breast cancer cells, suggesting a potential clinical therapeutical application of combination of EGCG with a histone deacetylase inhibitor in breast cancer. Our findings help to assess the key mechanisms of EGCG chemoprevention and therapy by impacting epigenetic pathways. Moreover, it will open new avenues to manage a subset of estrogen-resistant breast cancers and improve the survival rate in breast cancer by using these compounds, especially in combination.
Materials and methods
Cell culture and cell treatment
The ERα-positive MCF-7 and ERα-negative MDA-MB-231 breast cancer cell lines were obtained from American Type Culture Collection (ATCC). Cells were grown in phenol-red-free medium DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% dextran-charcoal-stripped fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and 1% penicillin/streptomycin (Mediatech, Herndon, VA). Cells were maintained in a humidified environment of 5% CO
2 and 95% air at 37°C. To evaluate ERα expression, MDA-MB-231 cells were treated with various concentrations of EGCG (Sigma, St. Louis, MO) for 3 days while MCF-7 cells served as a positive control. The medium with EGCG was replaced every 24 h for the duration of the experiment. Control cells received equal amounts of DMSO (Sigma) in the medium. For the combination study, cells were treated with an optimal concentration (10 μM) of EGCG based on our following results and 5-aza (2 μM for 2 days) (Sigma) or TSA (100 ng/ml for 12 h) (Sigma) alone or together for a total 3 days as the common recommended doses of these compounds [
12].
Trypan blue exclusion assay for cell viability
To determine the effects of ERα reactivation on cellular proliferation induced by EGCG, ERα-negative MDA-MB-231 and positive control MCF-7 cells were seeded in triplicate in 24-well plates. To determine the optimal concentration of EGCG on ERα expression, MDA-MB-231 cells were treated with various concentrations of EGCG. For the combination study, MDA-MB-231 cells were treated with 10 μM EGCG and 2 μM 5-aza or 100 ng/ml TSA alone or together for 3 days. To observe the effects of 17β-estradiol (E
2) (Sigma) and tamoxifen (Sigma) on ERα expression, EGCG and/or TSA-pretreated MDA-MB-231 cells were then exposed with/without 10 nM of E
2 or 1 μM tamoxifen [
12] for an extra two days, respectively. To determine cell viability, cells were trypsinized and resuspended in PBS (Phosphate buffered saline) buffer. Equal volumes of Trypan blue (0.4%) and cell suspensions were mixed and incubated at room temperature for 10 min. Both viable (unstained) and nonviable (stained) cells were counted using a hemacytometer. The percentages of viable cells were calculated by the formula: Viable cells (%) = number of viable cells per ml of aliquot/number of total cells per ml of aliquot × 100.
Quantitative real-time PCR
Both ERα-positive MCF-7 and ERα-negative MDA-MB-231 cells were cultured and treated as described above. Total RNA was extracted using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Genes of interest were amplified using 5 μg of total RNA reverse transcribed to cDNA using the Superscript II kit (Invitrogen) with oligo-dT primer. In the real-time PCR step, PCR reactions were performed in triplicate with 1 μl cDNA per reaction and primers specific for ERα (Hs01046818_ml), progesterone receptor (PGR) (Hs01556702_ml) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Hs99999905_ml) provided by Inventoried Gene Assay Products (Applied Biosystems, Foster City, CA) using the Platinum Quantitative PCR Supermix-UDG (Invitrogen) in a Roche LC480 thermocycler. Thermal cycling was initiated at 94°C for 4 min followed by 35 cycles of PCR (94°C, 15 s; 60°C, 30 s). GAPDH was used as an endogenous control, and vehicle control was used as a calibrator. The relative changes of gene expression were calculated using the following formula: fold change in gene expression, 2-ΔΔCt = 2-{ΔCt (treated samples) - ΔCt (untreated control samples)}, where ΔCt = Ct (ERα or PGR) - Ct (GAPDH) and Ct represents threshold cycle number.
Western blot analysis
For western blot analysis, protein extracts were prepared by RIPA Lysis Buffer (Upstate Biotechnology, Charlottesville, VA) according to the manufacturer's protocol. Proteins (100 μg) were electrophoresed on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were probed with antibodies to ERα (6F11; NeoMarkers, Fremont, CA), HDAC1 (H11; Santa Cruz Biotechnology), p300 (C-20; Santa Cruz Biotechnology) and SUV39H1 (44.1; Santa Cruz Biotechnology), then each membrane was stripped and reprobed with GAPDH antibody (V-18, Santa Cruz Biotechnology) as loading control. Molecular weight markers were run on each gel to confirm the molecular size of the immunoreactive proteins. Immunoreactive bands were visualized using the enhanced chemiluminescence detection system (Santa Cruz Biotechnology) following the protocol of the manufacturer.
Chromatin Immunoprecipitation (ChIP) assay
MDA-MB-231 cells were treated with 10 μM EGCG and 100 μg/ml TSA alone or in combination for the indicated times. Approximately 2 × 10
6 cells were cross-linked with a 1% final concentration of formaldehyde (37%, Fisher Chemicals, Fairlawn, NJ) for 10 min at 37°C. ChIP assays were performed with the EZ-Chromatin Immunoprecipitation (EZ-ChIP™) assay kit according to the manufacturer's protocol (Upstate Biotechnology) as described previously [
20]. The epigenetic antibodies used in the ChIP assays were ChIP-validated acetyl-histone H3 (Upstate Biotechnology), acetyl-histone H3-Lys9 (H3K9) (Upstate Biotechnology), acetyl-histone H4 (Upstate Biotechnology), histone deacetylase1 (HDAC1) (Santa Cruz Biotechnology), p300 (Santa Cruz Biotechnology), SUV39H1 (Santa Cruz Biotechnology), dimethyl-histone H3-Lys4 (H3K4) (Upstate Biotechnology), trimethyl-histone H3-Lys9 (H3K9) (Upstate Biotechnology) and DNMT1 (Abcam, Cambridge, MA). The transcription factor antibodies in this study were E2F4 (RK-13; Santa Cruz Biotechnology) and Rb/p130 (C-20; Santa Cruz Biotechnology). ChIP-purified DNA was amplified by standard PCR using primers specific for the ERα promoter yielding a 150 bp fragment: sense, 5'-GAACCGTCCGCAGCTCAAGATC-3' and anti-sense, 5'-GTCTGACCGTAGACCTGCGCGTTG-3'. PCR amplification was performed using the 2 × PCR Master Mix (Promega, Madison, WI) and the reaction was initiated at 94°C for 4 min followed by 30 cycles of PCR (94°C, 30 s; 56°C, 30 s; 72°C, 1 min), and extended at 72°C for 5 min. After amplification, PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide fluorescence using Kodak 1D 3.6.1 image software (Eastman Kodak Company, Rochester, NY). Quantitative data were analyzed using the Sequence Detection System software version 2.1 (PE Applied Biosystems, Foster City, CA).
HDACs and HATs activity assay
Cultured MDA-MB-231 cells were harvested at the indicated time points as described above, and nuclear extracts were prepared with the nuclear extraction reagent (Pierce, Rockford, IL). The activities of histone deacetylases, HDACs (Active Motif, Carlsbad, CA), and histone acetyltransferases, HATs (Epigentek, Brooklyn, NY), were performed according to the manufacturer's protocols as reported previously [
21]. The enzymatic activities of HDACs and HATs were detected by a microplate reader at 450 nm.
Bisulfite sequencing analysis
The DNA methylation status of the ERα promoter was detected by sodium bisulfite methylation sequencing. Approximately 1 μg genomic DNA was treated with bisulfite following the manufacture's protocol (Human Genetic Signatures, Macquarie Park, Australia). Bisulfite-modified DNA was amplified using two primer sets spanning a region from -66 to +356 of the ERα core promoter. PCR amplifications were performed with primers sense, 5'-AGTATTTTT GTAATGTATAT-3', and antisense, 5'-TCCAAATAATAAAACACCTA-3'. PCR products were purified using a gel extraction kit (Qiagen) and were directly cloned in pGEM-T vector according to manufacturer's protocol of pGEM-T Easy Vector Systems (Promega). Purified plasmids were sequenced with sense primer on an automated DNA sequencer. Each sample was sequenced on more than five clones to determine the site-specific methylation changes in the ERα promoter region.
Statistical analyses
Data from Real-time PCR and luciferase assays were derived from at least three independent experiments. For quantification of ChIP products, Kodak 1D 3.6.1 image software was used. The protein levels were quantified by optical densitometry using ImageJ Software version 1.36b
http://rsb.info.nih.gov/ij/. Statistical significance between treatment and control groups was evaluated using Mann-Whitney U test.
P < 0.05 was considered significant.
Discussion
The intriguing effects of the bioactive botanic component of green tea, EGCG, on cancer chemoprevention and therapy have received considerable attention [
30]. Various molecular mechanisms have been proposed involving EGCG-induced inhibitory effects on many types of cancers including breast cancer. Clinical prognosis and subsequent therapeutical strategies of malignant breast cancer rely on the expression of an important growth factor receptor, the nuclear estrogen receptor α (ERα) [
1]. Therefore, ERα-positive breast cancer patients receiving standard endocrine therapy by the use of the anti-estrogen drug such as tamoxifen, will generally have a better prognosis [
4,
24]. However, ERα-negative tumors display resistance to anti-hormone therapy due to the lack of targeting-directed therapies and this form of tumor is more aggressive and renders a poorer prognosis [
23,
24]. Thus, new therapies or strategies for sensitization of ERα-negative tumors to endocrine treatment are urgently required.
In the present study, we provided evidence that EGCG can induce re-expression of endogenous estrogen receptor α (ERα) in ERα-negative MDA-MB-231 breast cancer cells. For the first time, our results clearly show that this functional ERα reactivation by EGCG treatment is at least partly regulated via epigenetic mechanisms, especially through chromatin remodeling. We also found that this effect was synergistically enhanced when EGCG was combined with the deacetylation inhibitor, TSA, indicating histone modification plays an important role in EGCG-induced ERα reactivation. Furthermore, EGCG was found to influence the assembling of transcription repressors complex in the promoter region of ERα leading to ERα re-expression in ERα-negative breast cancer cells. Therefore our results indicate that green tea EGCG can sensitize ERα-negative breast cancer cells to respond to conventional anti-hormone therapy through reactivating ERα, which could provide a new avenue for therapeutical strategies of hormone-resistant breast cancer.
A number of findings have demonstrated that epigenetic regulation is one of the most important molecular mechanisms that result in the absence of estrogen receptor α (ERα) in hormone-resistant breast cancer cells [
6‐
9]. Previous studies have shown that applying different epigenetic-related enzymatic inhibitors such as the HDAC inhibitor, TSA, and the DNMT1 inhibitor, 5-aza, can reactivate functional ER expression suggesting that epigenetic mechanisms play a crucial role in ER transcription regulation [
8]. Recently, extensive studies have focused on a dietary component, EGCG, the most abundant catechin in green tea beverages, in regard to its chemopreventive and anticancer properties. Various mechanisms have been demonstrated for the anticancer property of EGCG including inhibition of cellular oxidative stress, inhibition of angiogenesis, and regulation of signal transduction. However, Fang et al. have found that EGCG can inhibit DNMT activity directly and indirectly, thereby leading to demethylation and reactivation of methylation-silenced tumor suppressor genes such as
p16
INK4a
,
RARβ and
MGMT in human esophageal cells [
18,
19]. Moreover, our previous studies also showed that EGCG treatment can inhibit telomerase activity through epigenetic regulation of the
hTERT (human telomerase reverse transcriptase) gene [
17]. Taken together, these results indicate that EGCG may affect epigenetic control of transcription regulation in certain epigenetic-sensitive tumor-related key genes such as the estrogen receptor gene in breast cancer cells.
As shown in our current study, we observed a relatively low concentration of EGCG treatment could induce a pronounced ERα re-expression in ERα-negative breast cancer cells suggesting that EGCG can reactivate the estrogen signal pathways via regulating ERα re-expression. More importantly, administration of EGCG has shown a huge chemopreventive potential on hormone-resistant breast cancer simply by drinking green tea to maintain a low level of EGCG in serum [
31]. Epigenetic mechanisms play an important role in ERα regulation. However, we only observed a synergistic effect on reactivating functional ERα expression when EGCG was combined with the HDAC inhibitor, TSA, rather than with the DNMT inhibitor, 5-aza, indicating histone modification may play a more important role in EGCG-induced ERα reactivation than DNA methylation. This hypothesis has been confirmed by our results showing that various chromatin markers were dramatically altered in the ERα promoter by EGCG treatment accompanied by corresponding alterations in the activities of histone modification-related enzymes such as HDACs and HATs in ERα-negative breast cancer cells. Consistently, we did not find any changes of DNA methylation patterns in the ERα promoter and the protein level of DNMT1 by EGCG treatment. However, a decreased binding of DNMT1 in the ERα promoter by EGCG treatment shown in Figures
4A and
4B may better explain a minor increased ERα expression when EGCG was combined with 5-aza compared to treatment with EGCG or 5-aza alone as indicated in Figure
1E. Taken together, our results showing a minor role of DNA methylation in EGCG-induced ERα reactivation do not contradict the demethylation nature of EGCG in previous studies, but rather highlight the gene and site-specificity of EGCG treatment on global DNA methylation patterns [
32]. It also elicits an interesting possibility that consumption of green tea and cruciferous vegetables such as brocoli, which are abundant in natural EGCG and the deacetylation agent such as sulforaphane [
33,
34], respectively, may result in a better chemopreventive outcome of breast cancer based on our current studies.
Abundant evidence has shown that gene transcription in eukaryotic cells is strongly influenced by interaction between transcription factors regulation and the modification of chromatin in the promoter regions of certain genes. In particular, epigenetic-related enzymes can not only affect the chromatin state, but also further influence the accessibility of the transcriptional machinery, resulting in gene activation or repression [
26‐
28]. A transcriptional complex model has been reported involving ERα regulation in breast cancer cells by Macaluso et al [
29], we then tested this concept in ERα-negative breast cancer cells by the treatment with EGCG and TSA alone or together. Our results revealed that EGCG can affect the binding of a multimolecular repressor complex, Rb/p130-E2F4/5-HDAC1-DNMT1-SUV39H1, to the ERα promoter, leading to ERα reactivation (Figure
5). This result therefore provides a key mechanism that modulates a crosstalk of both genetic and epigenetic signal transduction in ERα expression by EGCG as well as EGCG combined with a HDAC inhibitor.
Collectively, our studies investigate the basic epigenetic mechanisms by which green tea EGCG induces functional ERα reactivation in ERα-negative breast cancer cells. We found a relatively low concentration of EGCG could re-sensitize hormone-resistant breast cancers cells to the hormone antagonist, tamoxifen, by re-expression of functional ERα in ERα-negative breast cancer cells. In addition, EGCG-induced chromatin remodeling and accompanied binding changes of the transcriptional complex in the ERα promoter contribute to ERα reactivation. More importantly, these aforementioned effects were consolidated by combining EGCG with the deacetylation inhibitor, TSA, suggesting chromatin modulation plays a crucial role in EGCG-induced ERα reactivation.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
The first author, YL, designed and conducted the experiments and drafted the manuscript. The second author, YYY, assisted with some experiments. The third author, SMM, designed the experiments and reviewed the manuscript critically. The corresponding author, TOT, revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.