Background
Breast cancer is the most common cancer and the second most common cause of cancer-related death among women in the United States [
1]. This disease is usually treated through surgery and/or radiotherapy, supported by adjuvant endocrine or chemo-therapy. Unfortunately, most tumors acquire resistance during classical treatments [
2]. Therefore, there is a need for developing novel therapeutics for breast cancer. Approximately 70% of breast cancers are estrogen receptor alpha (ERα)-positive [
3]. ERα plays an important role in these cancers via both ligand-dependent and -independent mechanisms [
4]. ERα is a member of the super family of nuclear receptors that function as transcription factors. In addition to estrogen-induced activation, it also interacts with growth factor pathways [
5]. The role of ERα in breast cancer development has been extensively investigated. Transient over-expression of ERα promotes cell survival and estrogen-independent growth [
6] whereas ERα knock-down induces cell apoptosis and growth inhibition [
7] in estrogen-responsive MCF-7 breast cancer cells. Recent research also indicates that estrogen-independent ERα signaling and its interaction with growth factor receptors contribute to endocrine resistance in breast cancer treatment [
8]. As such, ERα has become an important target in developing breast cancer therapies.
Withaferin A (WA) is a steroidal lactone occurring in
Withania somnifera that has shown cytotoxicity in a variety of tumor cell lines and in animal cancer models
in vivo without any noticeable systemic toxicity [
9]. The mechanism of its action is currently under extensive investigation. It has been demonstrated that WA has the ability to alter numerous cancer-associated growth factor receptors, kinases, and transcription factors. It is a potent inhibitor of nuclear factor-κB activation [
10], and angiogenesis [
11]. In prostate cancer cell lines, WA binds to Heat shock protein 90 (Hsp90) and inhibits its chaperone activity, resulting in Hsp90 client protein degradation and tumor inhibition [
12]. Recent research revealed that treatment with WA causes apoptosis and growth inhibition in both the ERα-negative, p53-mutant MDA-MB-231 and the ERα-positive, p53-wildtype MCF-7 breast cancer cell lines, but MCF-7 cells exhibit higher sensitivity to the apoptotic effect of WA [
13,
14]. The molecular mechanism underlying the anti-cancer effects of WA in breast cancer is not well defined.
We hypothesized that ERα and its associated molecular network such as REarranged during Transfection (RET) tyrosine kinase and p53 may be involved in the anti-cancer effects of WA in MCF-7 breast cancer cells. ERα and the tumor suppressor protein p53 exert opposing effects on breast cancer cell proliferation and apoptosis. ERα promotes proliferation of breast cancer cells whereas p53 induces growth inhibition and apoptosis [
7]. RET is over-expressed in breast cancer ERα-positive breast cancer, and its activation stimulates MCF-7 breast cancer cell proliferation, survival and scattering [
5,
15]. In the present study, we examined the effects of WA on MCF-7 cell proliferation, viability, cell cycle distribution, and apoptosis, and addressed whether ERα and its associated molecular network may in part mediate the anti-cancer effects of WA in MCF-7 breast cancer cells.
Methods
Cell Lines and Reagents
The MCF-7 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA) and cultured in RPMI-1640 containing 10% fetal bovine serum (FBS), 100 units/Ml penicillin, 100 μg/mL streptomycin, 100 μg/mL gentamycin, 2 mM glutamine, and 1 mM pyruvate. The WA was purchased from ChromaDex (Irvine, CA). The antibodies against p53, RET, p21, poly(ADP-ribose) polymerase (PARP), caspase-3, caspase-7, p38 mitogen-activated protein kinase (p38 MAPK), phospho-p38 MAPK, heat shock factor 1 (HSF1) were purchased from Cell Signaling Technology (Danvers, MA) and the antibodies against ERα and survivin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-β-actin antibody was from Millipore (Billerica, MA). The anti-annexin V-fluorescein isothiocyanate (FITC)-conjugated and propidium iodide (PI) were from BD Bioscience (Rockville, MD).
MTS assay
The effects of WA on MCF-7 cell viability was examined using 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay as previously described with minor modification [
16]. Cells were seeded in 96-well microtiter plates (1.0 × 10
3 per well) in 100 μL of growth media. Cells were allowed to attach overnight and then treated with increasing concentrations of WA (0, 0.156, 0.313, 0.625, 1.25, 2.5, 5 μM) for 72 h. Cisplatin was used as a positive control. 20 μL of MTS solution was added at 72 h of treatment. After 3 h of incubation at 37°C (5% CO2), absorbance was measured at 490 nm with a microplate reader. The half-maximal inhibitory concentration (IC50) was obtained from the MTS viability curves using GraphPad Prism 5. Experiments were carried out in triplicates and the viability was expressed as the ratio of the number of viable cells with WA treatment to that without treatment. Experiments were repeated and similar results were obtained.
Trypan Blue Exclusion Assay
Cell proliferation was evaluated using trypan blue exclusion assay as described before [
17] with minor modification. MCF-7 cells were plated into 6-well plates at 20, 000 cells/well. After an overnight incubation, cells were treated with the vehicle (0.125% DMSO) or different doses of WA (0, 0.5, 1, and 2.5 μM) for 24 h. Cells exposed to 0.2% Trypan blue were then counted in a hemocytometer, and cells stained with Trypan blue were excluded. Percentage of viable cells was calculated based on the ratio of viable cell to total cell population in each well. The proliferation rate was calculated based on the number of viable cells in WA-treated group versus vehicle-treated group. Experiments were repeated to confirm the accuracy of the results.
Annexin V-FITC/PI Flow Cytometry
The effects of WA on MCF-7 cell apoptosis was examine using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry as described before [
16]. Cell were treated with 0, 1, 2.5, 5 μM of WA for 24 h or with 2.5 μM of WA for 0, 6, 12 and 24 h. Detached and adherent cells were then collected and labeled for 15 min at room temperature with annexin V-FITC (1 μg/mL) and with PI (40 μg/mL) and immediately analyzed on a BD™LSRII flow cytometer (BD Biosciences) using BD FACSDiva6.0 software.
Cell Cycle Analysis
The effects of WA on cell cycle distribution was examined using PI flow cytometry as described before [
16] with minor modification. MCF-7 cells were seeded in complete medium (2 × 10
5 per 100-mm plate). After an overnight incubation, cells were treated with 0, 0.5, 1, 2.5 μM of WA for 24 h or with 2.5 μM of WA for 0, 6, 12 and 24 h. Detached and adherent cells were collected and fixed in 70% ethanol in phosphate-buffered saline (PBS) at -20°C overnight. Cells were then resuspended in PBS containing 40 μg/mL PI and 100 μg/mL RNAse and incubated for 30 min at 37°C in the dark. Samples were then analyzed on a on a BD™LSRII flow cytometer (BD Biosciences) using BD FACSDiva6.0 software.
Western Analysis
MCF-7 cells were treated with 0, 0.5, 1, 2.5, 5 μM of WA for 24 h or with 2.5 μM of WA for 0, 1, 3, 6, 12, 24 and 48 h. Detached and adherent cells were collected, and total proteins were extracted using radioimmuno-precipitation assay buffer (RIPA buffer; 20 mM Tris [pH 7.5], 150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 1 mM ethylenediaminetetra-acetic acid, and 0.1% SDS) containing a protease/phosphatase inhibitor cocktail (0.1 mg/ml PMSF, 30 μl/ml of aprotinin, 5 μg/ml of leupeptin, and 1 mM sodium orthovanadate; Sigma). Protein concentrations were determined using the BCA protein assay reagent kit (Pierce, Rockford, IL). Equal amount of proteins were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted onto nitrocellulose membranes (Hybond; Amersham, Piscataway, NJ). After blocking with 3% nonfat dry milk in PBS for 1 h at room temperature, blots were incubated with appropriate primary antibodies overnight at 4°C. Blots were then washed and incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h and protein bands were visualized using a chemiluminescence kit (Thermo Scientific, Rockford, IL). Next, blots were stripped and reprobed for β-actin. The expression level of each protein was normalized to the level of β-actin.
In order to examine whether WA down-regulates ERα protein at post-translational level, we treated MCF-7 cells with 100 μg/ml of protein synthesis inhibitor cycloheximide in the absence or presence of 2.5 μM WA for 1, 2, 4, or 6 h. Cells were collected at each time points and total proteins were isolated and subjected to Western analysis for ERα and β-actin.
To examine whether proteasomal degradation of ERα is involved in ERα protein down-regulation by WA, MCF-7 cells were pre-treated with 30 μM of proteasome inhibitor MG132 for 1 h before treatment with 2.5 μM WA for 6 h. Cells were then collected and proteins in triton-soluble or triton-insoluble fractions were isolated as described previously [
18]. Briefly, cells were washed with PBS and lysed in RIPA buffer for 10 min on ice. After the cell suspensions were centrifuged at 16, 000 ×
g for 15 min at 4°C, the supernatants were collected as the Triton-soluble fraction. The pellet (Triton-insoluble fraction) was lysed in 2% SDS in 50 mM Tris-HCl and boiled for 15 min. The protein concentration of each sample was determined using the BCA protein assay. Samples were subjected to Western analysis for ERα and β-actin.
Statistical Analysis
All data were analyzed using SPSS Version 17.0 software (SPSS, Inc.). ANOVA was used for comparison across treatment regimes. When an F test indicated statistical significance, post hoc analysis was made using the Tukey's honestly significant difference procedure. Significance was set at P < 0.05 for all comparisons.
Discussion
In this study, we aimed to examine the anti-cancer effects of WA in ERα-positive MCF-7 breast cancer cells and explore the role of ERα and pathways associated with it in mediating these effects. We found that WA has potent inhibitory effects on the protein expression of ERα as well as RET tyrosine kinase that interacts with ERα pathway. WA also strongly up-regulated the protein levels of p53 and its down-stream target p21. Furthermore, WA at 2.5 or 5 μM markedly decreased HSF1 and survivin protein levels. These molecular changes correlate well with WA-induced dose- and time-dependent alterations in cell viability and apoptosis, suggesting that these are key factors involved in the anti-cancer effects of WA.
Most of breast cancer cases are hormone-dependent and higher levels estrogen are linked to increased risk of breast cancer [
22]. ERα is a transcription factor that plays a key role in mediating estrogen signaling. Recent research revealed that ERα also plays a critical role in breast cancer progression via estrogen-independent mechanisms [
23]. Knockdown of ERα expression using siRNA for ERα has been found to inhibit cell proliferation and induce apoptosis in MCF-7 breast cancer cells [
7]. As such, ERα has become a molecular target in the development of therapeutics for breast cancer. In this study, WA treatment caused drastic decrease in ERα protein levels, which most likely contributed to growth inhibition and apoptosis.
RET is a receptor tyrosine kinase that mediates the proliferative and pro-survival effects of GDNF family growth factors in breast cancer. It is over-expressed in breast cancer, with higher levels in ERα-positive tumors as compared to ERα-negative tumors. Activation of RET stimulates MCF-7 breast cancer cell proliferation, survival and scattering [
5,
15]. RET pathway functionally interacts with ERα pathway [
5]. In the present study, we found that WA down-regulates RET protein levels in parallel with ERα depletion. Down-regulation of both ERα and RET pathways may thus provide more effective anti-tumor activity.
The tumor suppressor p53 exerts its anti-proliferative action by inducing reversible or irreversible cell cycle arrest or apoptosis [
24]. P53 can block cell cycle progression and/or induce apoptosis by transactivation of specific target genes, including p21Waf-1/Cip1 (p21), Gadd45, cyclin G and Bax [
25,
26]. Loss of p53 activity inhibits apoptosis and accelerates the appearance of tumors in transgenic mice [
27]. The p53 gene is mutated in approximately 20% of breast cancers. Thus, although the majority of breast cancers have wild-type p53, its anti-cancer function remains suppressed. Additionally, p21 is a critical cell cycle inhibitor in many cancer cells including MCF-7 cells [
28]. It has been found to be essential for G2/M cell cycle arrest upon DNA damage [
29]. Although its role in apoptosis is controversial in general, it has been reported to induce apoptosis in MCF-7 cells [
19,
30]. In the present study, we found that WA increased the protein levels of p53 and p21, suggesting that they are involved in the growth inhibition and pro-apoptotic effects of WA. The pattern of p21 correlated well with that of p53, indicating that p53 may play a role in p21 up-regulation by WA. An up-regulation of phospho-p38 MAKP was observed at 1 h and 3 h post WA treatment, preceding the up-regulation of p53. Since phopho-p38 MAPK has been demonstrated to up-regulate p53 expression [
31], and WA is able to induce apoptosis by activating p38 in leukemic cells [
32], it appears that the p38 pathway may be an up-stream pathway involved in the anti-cancer effects of WA in MCF-7 cells.
Accumulating evidence indicates that there is a cross-talk between pathways mediated by ERα and p53. ERα and p53 exert opposing effects on breast cancer cell proliferation [
7]. ERα binds directly to p53, leading to down-regulation of transcriptional activation or depression by p53. For example, wild-type p53 can be functionally inhibited by ERα leading to up-regulation of survivin and suppression of apoptosis [
7]. RNA interference-mediated knockdown of ERα insulted in reduced survivin expression and enhanced apoptosis in MCF-7 breast cancer cells [
7]. The depletion of ERα in combination with the up-regulation of p53 following WA treatment in MCF-7 cells may render the cells more sensitive to apoptosis. The fact that WA can also induce apoptosis, although not as effective, in MDA-MB-231 breast cancer cell line [[
14], and unpublished data from our lab] which is ERα negative and p53-mutant, suggests that WA targets multiple pathways in its anti-cancer function, and neither ERα nor wild-type p53 is indispensable for the anti-cancer effect of WA. However, it is reasonable to postulate that in ERα-positive and p53-wild-type breast cancer cells, depletion of ERα and up-regulation of p53 substantially contribute to the anti-cancer effects of WA.
HSF1, the transcription factor for heat shock proteins (HSP), has recently been shown as a facilitator of transformation in breast cancer [
33,
21]. HSF1 inactivation inhibits the progression of a wide spectrum of cancers [
34,
35]. As the central regulator of HSP expression, HSF1 is also a target in designing anti-breast cancer therapies. HSF1 is another up-stream regulator of survivin. Down-regulation of HSF1 leads to decreased survivin expression in breast cancer cells [
21]. In the present study, 2.5 or 5 μM of WA markedly decreased HSF1 and survivin expression at 24 h, suggesting that HSF1 may play a role in WA-induced apoptosis in part by regulating survivin expression. At lower concentrations or early time points, WA induced HSF1 phosphorylation, which is likely due to a transient defensive response of cells to the stress caused by WA.
In this study, WA induction of apoptosis in MCF-7 cells was confirmed by Annexin V assay as well as PARP cleavage and decreased expression of the pro-caspase3 and pro-caspase7. The amount of pro-caspase7 is much more abundant as compared to pro-caspase3 in MCF-7 cells, and this is in agreement with earlier reports [
36]. At 1 μM, WA induced mainly early apoptosis. At higher concentrations (2.5 and 5 μM), the number of early apoptotic cells decreased whereas more late apoptotic cells were detected. It appears that whether it is early apoptosis or late apoptosis primarily depends on the concentration of WA, as mainly late apoptosis was detected with 2.5 μM WA at 12 h when apoptosis became detectable. WA causes minimal necrosis in MCF-7 cells. The small number of cells gated to the necrosis fraction after treatment with WA is most likely artifact caused by trypsinization, because when we tried Annexin V experiments at 36 h after 2.5 μM WA treatment without trypsinization (cells were all detached by 36 h), nearly 100% of cells were gated to the last apoptosis fraction (data not shown).
A recently published paper by Hahm et al. [
37] also reported down-regulation of ERα and up-regulation of p53. However, they declared that WA-mediated decline in ERα protein level is caused by transcriptional repression, instead of proteasomal degradation. Our data showed that WA decreased ERα at post-translational level by inducing aggregation and proteasome-dependent degradation of ERα protein. The reason why they failed to see any effect of MG132 on WA-induced decline in ERα protein level is most likely that they only examined ERα protein in Triton-soluble fraction but not in Triton-insoluble fraction.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
XZ conceived the study, carried out the experiments, and wrote the manuscript. RM and AKS assisted XZ in carrying out the experiments. MSC participated in designing the experiments. All authors read and approved the final manuscript.