Activation of the steroid and xenobiotic receptor, SXR, induces apoptosis in breast cancer cells
- 5.8k Downloads
The steroid and xenobiotic receptor, SXR, is an orphan nuclear receptor that regulates metabolism of diverse dietary, endobiotic, and xenobiotic compounds. SXR is expressed at high levels in the liver and intestine, and at lower levels in breast and other tissues where its function was unknown. Since many breast cancer preventive and therapeutic compounds are SXR activators, we hypothesized that some beneficial effects of these compounds are mediated through SXR.
To test this hypothesis, we measured proliferation of breast cancer cells in response to SXR activators and evaluated consequent changes in the expression of genes critical for proliferation and cell-cycle control using quantitative RT-PCR and western blotting. Results were confirmed using siRNA-mediated gene knockdown. Statistical analysis was by t-test or ANOVA and a P value ≤ 0.05 was considered to be significant.
Many structurally and functionally distinct SXR activators inhibited the proliferation of MCF-7 and ZR-75-1 breast cancer cells by inducing cell cycle arrest at the G1/S phase followed by apoptosis. Decreased growth in response to SXR activation was associated with stabilization of p53 and up-regulation of cell cycle regulatory and pro-apoptotic genes such as p21, PUMA and BAX. These gene expression changes were preceded by an increase in inducible nitric oxide synthase and nitric oxide in these cells. Inhibition of iNOS blocked the induction of p53. p53 knockdown inhibited up-regulation of p21 and BAX. We infer that NO is required for p53 induction and that p53 is required for up-regulation of cell cycle regulatory and apoptotic genes in this system. SXR activator-induced increases in iNOS levels were inhibited by siRNA-mediated knockdown of SXR, indicating that SXR activation is necessary for subsequent regulation of iNOS expression.
We conclude that activation of SXR is anti-proliferative in p53 wild type breast cancer cells and that this effect is mechanistically dependent upon the local production of NO and NO-dependent up-regulation of p53. These findings reveal a novel biological function for SXR and suggest that a subset of SXR activators may function as effective therapeutic and chemo-preventative agents for certain types of breast cancers.
KeywordsNitric Oxide Breast Cancer Cell Tamoxifen Rifampicin Breast Cancer Cell Line
Inducible Nitric Oxide Synthase
Nitric Oxide, QRT-PCR: Quantitative Real Time RT-PCR
Enzyme-Linked ImmunoSorbent Assay
Reactive Oxygen Species
Reactive Nitrogen Species
Anti-estrogens such as tamoxifen are important therapeutic agents in the treatment and chemoprevention of breast cancer . Other compounds such as fatty acid amides and retinoid × receptor (RXR) agonists are also effective against breast cancer in cell lines and in animal models [2, 3]. Interestingly, a macrolide antibiotic-rifampicin, the antimycotic drug clotrimazole, endogenous cannabinoids such as anandamide, RXR agonists (rexinoids) such as targretin, and tocotrienol forms of Vitamin E share the ability to inhibit the growth of various types of cancer [2, 3, 4, 5, 6, 7, 8]. Some of these compounds such as rifampicin, targretin, and tocotrienols have also been shown to have synergistic or additive effects against cancer when used together with tamoxifen [4, 7, 9]. Although it is certainly possible that they act through separate and distinct pathways, the ability of these structurally and functionally distinct compounds to inhibit the growth of cancer cells and their additive effects with tamoxifen raise the possibility that they might also act through a common molecular target. Notably, these compounds (including tamoxifen) share the ability to activate a heterodimer of the steroid and xenobiotic receptor (SXR , also known as PXR , PAR , and NR1I2 ) and retinoid × receptor (RXR) .
SXR is an orphan nuclear receptor activated by a large number of endogenous steroids and bile acids, prescription drugs, dietary compounds and xenobiotic compounds [15, 16, 17, 18]. SXR is highly expressed in the liver and intestine where it acts as a broad-specificity chemical sensor. SXR activation induces expression of genes involved in all three phases of drug and xenobiotic metabolism: hydroxylation by cytochrome P450 enzymes, conjugation to glutathione, sulfates and sugars, and transport by ABC family transporters (reviewed in [19, 20, 21]). We and others have shown that SXR is also expressed in tissues such as bone, kidney, lung, endometrium, and breast [22, 23, 24, 25, 26], where it may play roles other than its conventional role in metabolism. For example, activation of SXR in bone is associated with increased expression of bone biomarkers involved in maintaining bone homeostasis [27, 28]. SXR activation in liver is associated with decreased fibrogenesis and increased expression of anti-apoptotic genes such as BCL2 and BCL-XL [29, 30, 31]. Expression of SXR in endometrial cancer tissues is associated with decreased sensitivity to anti-cancer agents [22, 32]. Recently, one report has shown that activation of SXR leads to heightened sensitivity to oxidative cellular damage and apoptosis in transgenic mice and in cancer cells . Another suggested an anti-apoptotic role for SXR in colon cancer cells and in normal mouse colon epithelium 
SXR is expressed in breast cancer cells and tissues [23, 25]. The expression of SXR in breast cancer cells and tissues and the ability of numerous compounds active against breast cancer to activate SXR led us to hypothesize that SXR might serve as a common molecular target for their action. Here we show that SXR activation can inhibit breast cancer cell growth by inducing cell cycle arrest and apoptosis. We define a molecular pathway wherein activation of SXR inhibits proliferation of estrogen receptor positive (ER+) and p53 wild type (p53wt) breast cancer cell lines (MCF-7 and ZR-75-1) via induction of inducible nitric oxide synthase (iNOS), increased expression of nitric oxide (NO), and NO-dependent stabilization and accumulation of p53. SXR-mediated stabilization of p53 protein and up-regulation of p53 mRNA leads to increased expression of p53-dependent cell cycle regulators and pro-apoptotic genes such as p21, BAX and PUMA. Gain- and loss-of-function studies confirm that SXR is the mediator of this pathway. We conclude that activation of SXR is anti-proliferative in MCF-7 and ZR-75-1 breast cancer cells and that these effects are mediated through a NO and p53-dependent pathway. Our results identify a novel molecular pathway for SXR action in breast cancer and widen the biological relevance of SXR beyond xenobiotic metabolism.
Rifampicin, anandamide and camptothecin were from BioMol (Plymouth Meeting, PA) and all other compounds were from Sigma. Compounds were freshly diluted in DMSO prior to addition to cell culture media. The final DMSO concentration was < 0.05%. Breast cancer cell lines were maintained in phenol red free Improved Minimum Essential Media (IMEM; Mediatech, Kansas City, MO) supplemented with 10% fetal bovine serum (FBS; Invitrogen Corporation) (IMEM/FBS). LS180 cells were maintained in Dulbecco Modified Eagle Medium (DMEM; Cellgro) supplemented with 10% FBS.
Quantitative real-time RT-PCR
Primer sequences used in Q-RTPCR analysis
Cells growing in culture were washed three times with ice-cold PBS and then lysed in RIPA buffer (137 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1% Triton X-100, 0.5% NP-40, 10% glycerol, 2 mM EDTA, pH 8.0) plus protease inhibitors (Roche). Lysate protein concentrations were determined using the Bradford method and equal amounts of protein were separated on 8–10% SDS-polyacrylamide gels. For CaM protein, the proteins were separated on 4–20% gradient Tris-HCl gels. Proteins were transferred to Immobilon membrane (Millipore) using the semi-dry method (BioRad, Hercules, CA). Membranes were blocked for 1 hr with 5% non-fat dried milk in TBST (25 mM Tris-HCl pH 7.4, 135 mM NaCl, 2.5 mM KCl, 0.1% Tween-20) and then incubated overnight at 4°C with SXR (Anti-412, or PP-H4417, Perseus Proteomics inc., Japan) or p53 (FL-393 HRP, Santa Cruz Biotechnology Inc., USA) antibodies. The membranes were fixed in 0.2% glutaraldehyde in TBS for CaM protein before blocking in 3% BSA overnight followed by 1 hr incubation in mouse monoclonal CaM antibody (C-7055, Sigma-Aldrich, USA) at RT. The anti-412 SXR antibody was raised against a synthetic peptide sequence starting at amino acid 412 (CLRIQDIHPFATPLMQE), by Bethyl Laboratories, Inc., Montgomery, TX. Isolated IgG was affinity purified against this peptide prior to use. The primary antibody incubation was followed by 1 hr incubation at room temperature with HRP-conjugated secondary antibody (1:10,000; Santa Cruz Biotechnology Inc., USA). The bands were detected using the ECL Plus Western Blotting Detection System (Amersham Bioscience, USA). Chemiluminescence was assayed using an Alpha Innotech Fluorchem SP imager (Alpha Innotech Inc., CA, USA) and analyzed by densitometry using FluorChem AlphaEase FC software (Alpha Innotech).
MCF-7 cells were seeded at 500 cells/well and ZR-75-1 cells were seeded at 2500 cells/well in 96-well plates in IMEM supplemented with 10% FBS. Cells were treated with ligands at the indicated concentrations every other day for seven days. Cell proliferation was measured using a fluorescence assay (CyQuant, Molecular Probes) and a Cytofluor 4000 Fluorescence Multi-Well Plate Reader (PerSeptive Biosystems).
MCF-7 cells were incubated with 10 μM of SXR agonists (rifampicin, tamoxifen, anandamide, clotrimazole, RU486) or DMSO solvent control for 24 h. Cells were subsequently harvested using trypsin (0.5% w/v), centrifuged (140 g for 12 min), washed with PBS, and fixed with 2 ml ice-cold 70% ethanol overnight. Cells were centrifuged and resuspended with 10 μg/ml Propidium Iodide and 100 μg/ml RNase A at 37°C for 30 min. Samples were acquired on a FACSCalibur (BD Biosciences), and data were analyzed by CellQuest (BD Biosciences) and FlowJo (Tree Star, San Carlos, CA) software. Modfit (Verity Software, Topsham, ME) software was used to quantitate cell cycle using the fluorescence values of the FL2-area channel.
Detection of apoptosis
MCF-7 cells growing in culture were treated with 10 μM SXR ligands for 24, 48 and 72 hrs. Cells were also treated with 10 μM camptothecin for 24 hrs as a positive control for apoptosis. After the indicated period of treatment, cells were trypsinized and counted. Apoptosis was measured in the cytoplasmic fraction of equal numbers of cells using the Cell Death Detection ELISA (Roche Applied Science, Germany) using the manufacturer's recommended protocol.
NOS activity in cell lysates
MCF-7 cells were treated with 10 μM rifampicin or DMSO for indicated time periods. The cells were also treated with a cocktail of IL-1β (20 ng/ml), TNFα (15 ng/ml) and LPS (1 mg/ml) as a positive control for iNOS induction. After the indicated time periods, cell lysates were made in 1× homogenization buffer (25 mM Tris-Hcl pH7.4, 1 mM EDTA, 1 mM EGTA) by sonicating cells twice for 10 s at 10 amp. NOS activity was detected by measuring the conversion of 14C L-arginine (Amersham Bioscience, USA) into 14C L-citrulline by using nitric oxide synthase assay kit (Calbiochem Inc., USA). Radioactivity was measured by liquid scintillation counting and normalized to protein content.
Nitrite concentration in culture medium
NO production was measured as nitrite concentration in cell culture supernatants using the Griess method . Briefly, aliquots of media were removed from cells growing in culture in the presence or absence of SXR activators, followed by centrifugation to remove cells. In the cell-free supernatants nitrate, a stable metabolite of NO, was reduced to nitrite by incubating sample aliquots for 15 min at 37°C in the presence of 0.1 U/ml nitrate reductase (from Aspergillus species, Roche), 50 μM NADPH (Sigma, USA) and 5 μM FAD (Sigma, USA). When nitrate reduction was complete, NADPH was oxidized to avoid interference with subsequent nitrite determination. For this purpose, the reduced samples were incubated with 10 U/ml L-lactate dehydrogenase (from rabbit muscle) and 10 mM sodium pyruvate for 5 min at 37°C, followed by addition of 50 μl of 1% sulfanilic acid in 5% phosphoric acid and 50 μl of 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride to 100 μl of the reduced cell-free supernatant . After 10 minutes at 23°C, the absorbance at 546 nM was determined. Concentrations of nitrite in samples were calculated from standard curves using sodium nitrite as the reference compound.
Small interfering RNA (siRNA) duplexes targeting human SXR and p53 were custom synthesized by Qiagen (Qiagen Inc. USA). The most effective SXR siRNA target sequence was 5'-GGCCACTGGCTATCACTTC-3' [27, 37] and p53 siRNA sequence was 5'-AAACCACTGGATGGAGAATATTT-3'. Non-silencing control siRNA sequence 5'-AATTCTCCGAACGTGTCACGT-3' (Qiagen Inc. USA) was used as a negative control. The cells were transfected using Lipofectamine™ RNAiMAX transfection reagent (Invitrogen Inc., USA). The knock down efficiency by mRNA and protein were determined after 48 and 72 hrs of transfection, respectively. For gene expression assays, the cells were transfected with siRNA two day prior to ligand treatment, or the day of ligand treatment. After 48 hr of treatment with SXR ligands, RNA was isolated, cDNA synthesized and QRT-PCR analysis performed.
Data are shown as the mean values ± SEM. Results of two groups were analyzed using "unpaired" Student's t tests. Multiple comparisons were analyzed using one-way analysis of variance (ANOVAs). P ≤ 0.05 was taken to be statistically significant.
SXR mRNA and protein are expressed in breast cancer cell lines
Considering the relatively low levels of SXR mRNA found in breast cancer cells, western blot analysis of cell lysates was employed to confirm that SXR protein was, in fact, expressed. In agreement with the QRT-PCR data, SXR protein was present in both breast cancer cell lines (for characterization of the antibody, see Additional file 1: supplementary Figure 1). Consistent with these findings, MCF-7 and ZR-75-1 cells treated with 10 μM of the SXR activators rifampicin, anandamide or clotrimazole for 48 hours showed induction of the well-characterized SXR target gene CYP3A4 by QRT-PCR, demonstrating the functionality of endogenous SXR in these cells (data not shown). The kinetics of CYP3A4 up-regulation (48 hour post-treatment) by SXR activators matches with previously reported timing for its induction by SXR activators in osteosarcoma cells , where its activation induces genes involved in bone homeostasis.
SXR activation reduces the proliferation of breast cancer cell lines
To determine the dose dependent effect of SXR activators on proliferation, breast cancer cells were cultured in the presence of a concentration series (1, 10 and 100 μM) of test compounds or controls (solvent, rutin). To measure the integrated effect on cell cycle and/or cell survival in the presence of SXR activators the proliferation assays were continued until 7 days which was the time when the cells in the solvent control wells just reached confluency. Interestingly, SXR activators elicited a dose-dependent reduction in proliferation of both cell types (Figure 2A). The rank order of potency in the proliferation assay agreed well with the potency and efficacy of these compounds as SXR activators. For example, rifampicin, which was the best activator of SXR in transfection assays, also had the highest ability to inhibit proliferation of breast cancer cells in proliferation assay. Rutin, which did not activate SXR in transfection assays, lacked any effect on proliferation of cancer cells (Figure 2A and Additional file 1: supplementary Figure 2). The anti-proliferative effects of tamoxifen were more pronounced than its ability to activate SXR, but, as both of the tested breast cancer cell lines are estrogen receptor positive, it is likely that some of the observed effects are mediated through an ER-dependent mechanism.
SXR activation induces apoptosis and cell cycle arrest in breast cancer cells
The decreased proliferation observed in breast cancer cell lines treated with SXR activators could be due to inhibition of proliferation, increased cell death or both. To determine the earlier molecular events responsible for the ultimate effect on cell number, we first examined the effects of SXR activator treatment on the cell cycle using flow cytometry. In proliferation assays, the growth inhibitory effects of SXR activators were obvious at doses as low as 10 μM (Figure 2A). Therefore, we chose a 10 μM dose of SXR activator compounds for all further experiments. MCF-7 cells were treated with SXR activators rifampicin, tamoxifen, anandamide, clotrimazole, RU486  or solvent control for a time course of 12, 24 and 48 hrs so that the earlier molecular events can be detected within one or two cell cycles. Cellular DNA content was measured by propidium iodide staining followed by FACS analysis. All SXR activator treated MCF-7 cells exhibited a consistent increase in the fraction of cells in the G0/G1 phase of the cell cycle compared with controls starting at 24 hours, suggesting that they are arrested at G0/G1 (Figure 2B). The cells treated with SXR activators accumulated more in G1/S phase at 48 hr but the cells in solvent control also increased in G1/S phase at 48 hr time point, suggesting that the solvent control cells have started to get confluent at that time. The SXR activators consistently had more cells in G1/S phase than did solvent controls, suggesting that SXR activators can specifically cause G1/S arrest in breast cancer cells (data not shown).
This finding is consistent with published results showing that tamoxifen (an anti-estrogen and SXR activator) causes G0/G1 arrest in MCF-7 cells . Although tamoxifen, RU486 and nifedepine can activate SXR, they can also affect ER and PR/GR and calcium homeostasis of the cells respectively, which can have erroneous effects on cell growth. To avoid unnecessarily confounding the results, we decided to use only the antibiotic rifampicin, the anti-fungal clotrimazole and the fatty acid amide anandamide as SXR activators in subsequent experiments.
We next tested the effect of SXR activation on apoptosis using a sandwich ELISA with anti-histone and anti-DNA antibodies. Again MCF-7 cells were treated with 10 μM of the SXR activators rifampicin, anandamide, clotrimazole or solvent from 24–72 hours, or treated with 10 μM camptothecin for 24 hours as a positive control for induction of apoptosis. Each of the SXR activators increased the amount of apoptosis observed in treated MCF-7 cells beginning at 48 hours (Figure 2C). Therefore, we infer that the decreased proliferation observed in MCF-7 cells is caused by an increase in cell cycle arrest at G0/G1 phase followed by apoptosis.
Treatment with SXR activators increases expression of p53 and p53 target genes
We also tested the effects of SXR agonists on steady state levels of p53 protein in MCF-7 and ZR-75-1 cells. Camptothecin treatment for 24 hrs served as a positive control for p53 induction. Treatment with each of the SXR activators resulted in increased p53 protein levels in both MCF-7 (Figure 3C) and ZR-75-1 (Figure 3D) cells. Previous reports showed that iNOS-mediated increases in nitric oxide (NO) levels lead to p53 stabilization and activation as a result of phosphorylation of p53 at ser-15 . This phospho-p53 is transcriptionally active and increases expression of p21 and BAX [50, 51]. Since iNOS is a reported target gene of ligand-activated SXR , we further investigated the idea that the mechanism linking SXR activation with the observed effects on p53 and its target genes involved iNOS expression and increased NO levels.
Inducible nitric oxide synthase expression and nitric oxide levels are increased by SXR activation
Under normal conditions, calmodulin (CaM) binding is necessary for the stabilization and activation of iNOS. When CaM levels are insufficient, newly synthesized iNOS is rapidly degraded through the calpain and proteasomal systems . Therefore, we tested the expression of CaM mRNA in MCF-7 cells treated with rifampicin or solvent control for 3 hrs, 9 hrs, 12 hrs and 24 hrs and ZR75-1 cells treated with rifampicin or solvent control for 3 hrs, 9 hrs, 12 hrs and 18 hrs using QRT-PCR. We observed a transient up-regulation in steady state levels of CaM mRNA at 9 hrs in MCF-7 cells and at 18 hours in ZR75-1 cells (see Additional file 1: supplementary Figure 3A and 3B). To further confirm that this transient increase in CaM mRNA translated into sustained increase in protein level we tested the protein expression of CaM in MCF-7 and ZR-75-1 cells treated with rifampicin, anandamide or solvent control for 24 hrs and 18 hrs respectively. These timings correspond with when we noted up-regulation of iNOS mRNA in these cell lines. As shown in Figure 4C, CaM protein was up-regulated in comparison to the solvent control, DMSO by both SXR activators in both MCF-7 and ZR-75-1 cells. This is in accord with the notion that increased iNOS activity requires increased CaM to stabilize the newly synthesized iNOS . In MCF-7 cells CaM mRNA is down by 24 hrs but CaM protein was still up till 24 hrs by both SXR activator compounds, rifampicin and anandamide suggesting long half-life of the protein.
Two approaches were employed to confirm that the elevated levels of iNOS correspond with increased NOS activity. First, we tested NOS activity in homogenates made from MCF-7 cells treated with 10 μM rifampicin or solvent control for 24 and 48 hours by measuring in-vitro biochemical conversion of 14C L-arginine into 14C L-citrulline as a direct measure for iNOS activity . Second, we determined the NO concentration in cell free supernatant from SXR activator treated MCF-7 cells by measuring the total nitrate plus nitrite (the stable metabolites of NO) present in the media as an indirect measure of iNOS activity by the Greiss method . Treatment of MCF-7 cells with rifampicin elicited a significant increase in NOS activity (Figure 4D). Treatment with a cytokine cocktail was used as a positive control for iNOS induction . The increased NOS activity by either rifampicin or by cytokine cocktail treatment could be completely blocked by either a non-specific NOS inhibitor, L-N(G)-monomethylarginine (L-NMMA)  or the iNOS specific inhibitor 1400 W  (Figure 4D). Together, these results confirm that the increase in NOS activity in MCF7 cells resulting from treatment with SXR activators is primarily due to increased iNOS levels and activity.
Consistent with increased iNOS expression and NOS activity, the total nitric oxide level in the culture medium of MCF-7 cells treated with 10 μM rifampicin, anandamide, or clotrimazole for 48 hours were also increased (Figure 4E), confirming that iNOS-dependent increased production of NO could be one important mechanism through which SXR activators promote apoptosis in these cells. As expected based on the NOS activity, NO production was also blocked in SXR activated cells using the inhibitors 1400 W or L-NMMA (see Additional file 1: supplementary Figure 3C).
To assess whether SXR activation also affected the expression of eNOS (endothelial NOS), MCF-7 and ZR-75-1 cells were treated with 10 μM rifampicin for 24 and 18 hrs respectively. Treatment with SXR activators selectively increased the expression level of iNOS but not of eNOS in both breast cancer cell lines (see Additional file 1: supplementary Figure 3D). These results further support that observed increase in NOS activity and NO by SXR activators is primarily due to increased levels of iNOS.
Since increased NO levels have been reported to lead to p53 stabilization and activation, we next studied the requirement for NO in the up-regulation of p53 and subsequent cell cycle arrest and apoptosis that we observed in breast cancer cells. We treated MCF-7 cells with rifampicin in the presence or absence of L-NMMA or 1400 W, followed by QRT-PCR to measure the expression level of p53. Pre-treatment of MCF-7 cells with L-NMMA or 1400 W completely blocked the up-regulation of p53 observed following rifampicin treatment (Figure 4F). Therefore, we infer that increased cellular NO levels are required for up-regulation of p53 and its target genes in response to SXR activation.
Treatment with 1400 W itself activated p53 expression in breast cancer cells (Figure 4F). Since many known drugs activate SXR, we tested the ability of L-NMMA and 1400 W to activate SXR by measuring levels of the SXR target gene CYP3A4. Treatment with 1400 W, but not L-NMMA, increased CYP3A4 levels. We concluded that 1400 W activates SXR, which might explain its ability to up-regulate p53 (see Additional file 1: supplementary Figure 3E). Even though 1400 W can activate p53 on its own it was still able to inhibit rifampicin induced p53 up-regulation (Figure 4F). One possible explanation is that rifampicin is a higher affinity ligand for SXR than is 1400 W. Thus, in the absence of rifampicin, 1400 W can bind to and activate SXR. However, in the presence of rifampicin, the 1400 W is competed out and functions only as an iNOS inhibitor. Although this hypothesis needs further exploration, but since both of the NOS inhibitors (L-NMMA and 1400 W) were able to inhibit p53 up-regulation by SXR activators, we infer that that the SXR activator-induced increase in p53 expression is mediated through iNOS and the up-regulation of p53 by 1400 W does not affect our conclusion.
Activity of SXR is sufficient to inhibit the growth of MCF-7 cells
We further confirmed our model by testing the gene expression level of iNOS and p53 and p53 dependent target genes in VP16 or VP16-SXR transfected MCF-7 cells. The expression of iNOS and p53 and p53 dependent genes was strongly induced in VP16-SXR transfected cells in comparison to VP16 alone transfected cells (see Additional file 1: supplementary Figure 4A). This further supports our model that the SXR activator-dependent effects on proliferation of breast cancer cells are mediated specifically through SXR-induced increase in iNOS and p53 and p53 dependent genes and not because of other properties of the compounds.
SXR knockdown by siRNA inhibits the increased expression of iNOS
As a final confirmation of the requirement for SXR in the regulation of iNOS expression and iNOS-mediated up-regulation of p53 and its target genes in breast cancer cells, we investigated the effects of SXR loss-of-function on the induction of gene expression by SXR ligands. MCF-7 cells were transfected with scrambled or SXR specific siRNA [27, 37]. Consistent with previously published reports treatment with a specific siRNA duplex against SXR, but not with a control scrambled siRNA, reduced the SXR mRNA level by more than 80% (see Additional file 1: supplementary Figure 4B), and protein levels by more than 50% in MCF-7 cells (Figure 5B). Rifampicin and anandamide induced up-regulation of both CYP3A4 and iNOS mRNA expression was also significantly inhibited by the SXR siRNA in these cells (Figure 5C). Similarly the SXR activator induced expression of p53 and p21 was also significantly inhibited by siRNA (see Additional file 1: supplementary Figure 4C).
As a final confirmation of the functional impact of SXR knockdown, we measured apoptosis in SXR siRNA treated cells. MCF-7 cells were transfected with scrambled or SXR specific siRNA. 36 hours after transfection, the cells were treated with rifampicin or solvent control. 72 hr post-treatment apoptosis was measured in equal number of cells using cell death detection ELISA. As shown in Figure 5D the un-transfected and SCR transfected cells showed a significant increase in apoptosis after rifampicin treatment compared with controls. In contrast, there was no induction of apoptosis by rifampicin in MCF-7 cells transfected with SXR-siRNA (Figure 5D). These results confirm the requirement for SXR function to mediate the iNOS and p53-dependent downstream pathways resulting in apoptosis and cell cycle arrest described above.
Knockdown of p53 inhibits up-regulation of p21 and BAX
The orphan nuclear receptor SXR is known to regulate the expression of target genes involved in all three phases of steroid and xenobiotic metabolism in the liver and gut. However, SXR is also expressed in bone, kidney, lung, endometrium and breast tissue [22, 24, 25, 26, 39]. Most of these tissues do not contribute significantly to xenobiotic metabolism; hence, SXR may be performing other functions. Here we have defined a new mechanism by which SXR transcriptional activation affects the growth of breast cancer cells. Activation of SXR in ER+p53wt breast cancer cells inhibits their proliferation through a cascade of events beginning withinduction of iNOS and production of NO that ultimately leads to cell cycle arrest and apoptosis.
It was previously established that RNS/NO cause p53 stabilization and that the stabilized p53 is transcriptionally active . NO-induced p53 stabilization is hypothesized to result from phosphorylation at serine-15 of p53 . This phosphorylated form of p53 has been associated with attenuated p53 nuclear export . In turn, nuclear levels of activated p53 are subject to negative regulation by Mdm2, which functionally inactivates p53, facilitates p53 ubiquitination, nuclear export, and proteasomal degradation. Whether or not phosphorylation at serine 15 disrupts the p53/Mdm2 interaction is currently controversial . However, it is clear that blocking p53 nuclear export stabilizes active p53  and that NO exposure leads to nuclear p53 accumulation . Moreover, expression of the iNOS gene has been shown to have a strong association with p53 expression at both mRNA and protein levels . Increased BAX, PUMA and p53 mRNA levels were not observed by all three SXR activators until 72 hours in MCF-7 cells, whereas iNOS was up-regulated as early as 24 hrs after treatment with SXR activators in our experiments (Figure 3A and Figure 4A). This timing supports our model that the primary effect of SXR activation in these breast cancer cells is to increase steady-state levels of iNOS mRNA which then leads to increased production of NO. Increased NO levels up-regulate expression of p53. The elevated levels of p53 further up-regulate expression of p21 and that of the pro-apoptotic p53 target genes BAX and PUMA in breast cancer cells (Figure 7). Inhibition of NOS blocked the induction of p53 and knock down of p53 inhibited the induction of p21 and BAX in response to SXR activators. Both of these results further confirmed our model that p53 is acting down-stream of NOS and is required for up-regulating the expression of cell cycle regulatory and pro-apoptotic genes such as p21, BAX and PUMA in breast cancer cells in response to SXR activation.
Interestingly, expression of the cell cycle regulatory gene p21(Waf1/Cip1) can also be induced by NO [50, 67]. Moreover, nitric oxide can have cytostatic effects on cells by directly influencing the cyclin D1 expression, independent of p53 . In our experiments MCF-7 accumulated in G1/S phase of cell cycle starting as early as 24 hours post-treatment. In contrast, p53 and its target genes did not started going up until 48 hours, indicating p53 independent cytostatic effects of nitric oxide at earlier time points.
Experiments in p53 null mice as well as in p53wt versus p53mut lymphoblastoid cells have demonstrated that NO induces apoptosis, and that this effect is dependent upon the presence of wild type p53 . We detected elevated NO and apoptosis in MCF-7 cells treated with SXR activators, and increases in iNOS mRNA as well as mRNAs encoding p53 and p53-target genes in MCF-7 and ZR75-1 cell lines. Both of these cell lines are p53 wild type; therefore, our results are consistent with the earlier findings. As expected, we did not observe the induction of p53 target genes by SXR activators in cells transfected with p53 siRNA which again emphasize the role of p53 in our model.
Previously published reports have suggested that there is an inverse relationship between ER and SXR levels in breast and endometrial tissues [22, 25]. Both of the cell lines tested in this study are ER positive. However, in our preliminary experiments with ER+ and ER- breast cancer cell lines and consistent with previously published reports with breast cancer cell lines , we did not observe an inverse relationship between ER and SXR protein levels (data not shown). It is possible that this may be because insufficient samples have been examined and this point requires further exploration with bigger sample size. It would also be of interest to see if the anti-proliferative effects of SXR are limited to ER+ breast cancer cells.
Interestingly, in this study we also found that although MCF-7 and ZR-75-1 cells express almost equal amount of SXR protein (Figure 1), the MCF-7 cells were considerably more sensitive in anti-proliferative response to SXR activators in comparison to ZR-75-1 cells (Figure 2A). There can be many reasons for differences in the sensitivity of these cells towards SXR activators. One plausible and likely explanation is that ZR-75-1 cells express much lower levels of p53 in comparison to MCF-7 cells http://www.mdanderson.org/departments/cancerbiology/dIndex.cfm?pn=31062032-B0EB-11D4-80FB00508B603A14. The topoisomerase I inhibitor, camptothecin can only induce a ~3 fold increase in p53 protein levels in ZR75-1 cells compared with ~18 fold increase in MCF-7 cells (please compare figure 3C and Figure 3D). This is consistent with the possibility that differences in the sensitivity of these cells to SXR activators is more closely related to the inducibility of p53, than to the levels of SXR.
A very recent study has shown that SXR activators increased the expression of organic anion transporter, enhancing estrogenic effects in breast cancer . The differences in the pro-proliferative vs. pro-apoptotic effects of SXR in this study vs. our study can have several possible causes. There was a different experimental design in that the other study grew the breast cancer cells in estrogenic conditions vs. our experiments in which the cells were grown in estrogen depleted conditions (stripped medium, phenol red free) or the choice of breast cancer cell lines (MCF-7 and ZR-75-1 vs. T47-D). In addition, MCF-7 cells express much lower level of OATP1a2 than T47-D and T47-D are p53mut.
Recent studies have reported anti-apoptotic effects of SXR in liver and colon cancer cells [29, 34] and proliferative effects in ovarian cancer cells in-vitro , whereas SXR activation, in-vivo, has been suggested to have pro-apoptotic effects in colon tissues . Here we have shown that activation of SXR is pro-apoptotic in breast cancer cells. The ability of SXR to be pro-apoptotic in one tissue and anti-apoptotic in others may result from cell-type specific effects of SXR, or of SXR-induced NO. We previously, showed that SXR can regulate gene expression in a tissue specific manner based on the levels of co-repressor NCoR expressed in these tissues . Therefore, we propose that SXR activation might activate a different panel of gene in liver or colon tissue in comparison to breast tissue which may explain its different role between tissues. Moreover, the association between RNS/NO and apoptosis is also very complex. RNS/NO can provoke apoptosis or alternatively antagonize cell death pathways, depending on the cell type, NO concentration, redox state of the cell, pro-versus anti-apoptotic balance of an individual cell, and the subcellular localization of NO synthesis (reviewed in ). Therefore, the threshold levels for RNS to trigger apoptosis will likely differ from one cell to another. Interestingly, in accordance with our results different groups have reported induction of NO by many different SXR activators such as rifampicin, tamoxifen and anandamide in different cell types [72, 73, 74]. These studies from different groups suggest that SXR might commonly activate NO in different cells but depending on the cellular milieu, SXR generated NO can be pro-apoptotic or anti-apoptotic. Taken together, these studies suggest that SXR may have cell type specific effects on proliferation/apoptosis pathways, particularly in cancer cells.
These results also raise an intriguing question – is SXR expression pro- or anti-proliferative in breast cancers, in vivo? Understanding whether SXR is pro- or anti-proliferative in breast cancers is very important for optimizing breast cancer therapies because many commonly used chemotherapeutic agents (tamoxifen, taxol, cyclophosphamide, cisplatin) are SXR activators. The novel link we have established between SXR and breast cancer requires further investigation using appropriate in vivo systems. If, as we have shown, SXR activation is anti-proliferative, then therapies or preventive measures that target SXR without inducing their own metabolism will provide an important adjunct to current therapies. If SXR expression promotes, rather than inhibits the growth of cancer cells, then treatment with drugs that activate SXR will ultimately be counterproductive to tumor treatment. In this event, treatment of patients with standard therapies together with SXR antagonists [28, 75], or with SXR-transparent chemotherapeutic agents could prove beneficial both in blocking tumor growth and in improving the therapeutic efficacy of existing agents.
Taken together, the data presented above show that activation of SXR induces apoptosis in p53wt breast cancer cells that is mechanistically dependent upon induction of iNOS and NO-induced accumulation of p53 in cells. Our results have established a novel link between SXR and p53 induction and apoptosis in breast cancer cells. There have been scattered reports on xenobiotic metabolism and breast cancer. However, it was not clear what pathways or genetic factors are involved. Our results represent the first identification of an association between a well established xenobiotic receptor, SXR and apoptosis of breast cancer cells. Further in depth studies providing mechanistic insights into the role of SXR in proliferation and apoptosis of mammary epithelial cells in-vivo will have significant impact on breast cancer.
Supported by grants from the NIH (CA087222), DOD (DAMD17-02-1-0323) and Avon Breast Cancer Foundation to BB. SV is the recipient of a predoctoral fellowship from the DOD (W81XWH-06-1-0453). MMT was supported by a training grant from the NIH (Ruth L. Kirschstein NRSA T32 HD-07029) and the California Breast Cancer Research Program (5FB-0100). We thank R. Brachmann for providing us with p53 siRNA. We also thank E. Lee, R. Kaigh, C. Walsh, R. Brachmann and members of Blumberg lab for constructive comments on the manuscript, P. Brown for stimulating discussions in the early phases of this work, and D. Fruman and C. Walsh for access to the FACSCalibur analyzer, software and help in data analysis. We thank A. Shah for help in conducting p53 experiments and A. Sun for early characterization of the anti-412 antiserum.
- 3.De Petrocellis L, Melck D, Palmisano A, Bisogno T, Laezza C, Bifulco M, Di Marzo V: The endogenous cannabinoid anandamide inhibits human breast cancer cell proliferation. Proceedings of the National Academy of Sciences of the United States of America. 1998, 95 (14): 8375-8380. 10.1073/pnas.95.14.8375.CrossRefPubMedPubMedCentralGoogle Scholar
- 12.Bertilsson G, Heidrich J, Svensson K, Asman M, Jendeberg L, Sydow-Backman M, Ohlsson R, Postlind H, Blomquist P, Berkenstam A: Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proceedings of the National Academy of Sciences of the United States of America. 1998, 95 (21): 12208-12213. 10.1073/pnas.95.21.12208.CrossRefPubMedPubMedCentralGoogle Scholar
- 14.Sane RS, Buckley DJ, Buckley AR, Nallani SC, Desai PB: Role of human pregnane × receptor in tamoxifen- and 4-hydroxytamoxifen-mediated CYP3A4 induction in primary human hepatocytes and LS174T cells. Drug metabolism and disposition: the biological fate of chemicals. 2008, 36 (5): 946-954. 10.1124/dmd.107.018598.CrossRefGoogle Scholar
- 15.Goodwin B, Gauthier KC, Umetani M, Watson MA, Lochansky MI, Collins JL, Leitersdorf E, Mangelsdorf DJ, Kliewer SA, Repa JJ: Identification of bile acid precursors as endogenous ligands for the nuclear xenobiotic pregnane × receptor. Proceedings of the National Academy of Sciences of the United States of America. 2003, 100 (1): 223-228. 10.1073/pnas.0237082100.CrossRefPubMedGoogle Scholar
- 16.Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT, Kliewer SA: The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. The Journal of clinical investigation. 1998, 102 (5): 1016-1023. 10.1172/JCI3703.CrossRefPubMedPubMedCentralGoogle Scholar
- 18.Moore LB, Goodwin B, Jones SA, Wisely GB, Serabjit-Singh CJ, Willson TM, Collins JL, Kliewer SA: St. John's wort induces hepatic drug metabolism through activation of the pregnane × receptor. Proceedings of the National Academy of Sciences of the United States of America. 2000, 97 (13): 7500-7502. 10.1073/pnas.130155097.CrossRefPubMedPubMedCentralGoogle Scholar
- 23.Miki Y, Suzuki T, Kitada K, Yabuki N, Shibuya R, Moriya T, Ishida T, Ohuchi N, Blumberg B, Sasano H: Expression of the steroid and xenobiotic receptor and its possible target gene, organic anion transporting polypeptide-A, in human breast carcinoma. Cancer research. 2006, 66 (1): 535-542. 10.1158/0008-5472.CAN-05-1070.CrossRefPubMedGoogle Scholar
- 24.Tabb MM, Sun A, Zhou C, Grun F, Errandi J, Romero K, Pham H, Inoue S, Mallick S, Lin M, et al: Vitamin K2 regulation of bone homeostasis is mediated by the steroid and xenobiotic receptor SXR. The Journal of biological chemistry. 2003, 278 (45): 43919-43927. 10.1074/jbc.M303136200.CrossRefPubMedGoogle Scholar
- 27.Ichikawa T, Horie-Inoue K, Ikeda K, Blumberg B, Inoue S: Steroid and xenobiotic receptor SXR mediates vitamin K2-activated transcription of extracellular matrix-related genes and collagen accumulation in osteoblastic cells. The Journal of biological chemistry. 2006, 281 (25): 16927-16934. 10.1074/jbc.M600896200.CrossRefPubMedGoogle Scholar
- 29.Zucchini N, de Sousa G, Bailly-Maitre B, Gugenheim J, Bars R, Lemaire G, Rahmani R: Regulation of Bcl-2 and Bcl-xL anti-apoptotic protein expression by nuclear receptor PXR in primary cultures of human and rat hepatocytes. Biochimica et biophysica acta. 2005, 1745 (1): 48-58.CrossRefPubMedGoogle Scholar
- 30.Haughton EL, Tucker SJ, Marek CJ, Durward E, Leel V, Bascal Z, Monaghan T, Koruth M, Collie-Duguid E, Mann DA, et al: Pregnane × Receptor Activators Inhibit Human Hepatic Stellate Cell Transdifferentiation In Vitro. Gastroenterology. 2006, 131 (1): 194-209. 10.1053/j.gastro.2006.04.012.CrossRefPubMedGoogle Scholar
- 31.Marek CJ, Tucker SJ, Konstantinou DK, Elrick LJ, Haefner D, Sigalas C, Murray GI, Goodwin B, Wright MC: Pregnenolone-16alpha-carbonitrile inhibits rodent liver fibrogenesis via PXR (pregnane × receptor)-dependent and PXR-independent mechanisms. The Biochemical journal. 2005, 387 (Pt 3): 601-608.CrossRefPubMedPubMedCentralGoogle Scholar
- 33.Gong H, Singh SV, Singh SP, Mu Y, Lee JH, Saini SP, Toma D, Ren S, Kagan VE, Day BW, et al: Orphan nuclear receptor pregnane × receptor sensitizes oxidative stress responses in transgenic mice and cancerous cells. Molecular endocrinology (Baltimore, Md). 2006, 20 (2): 279-290. 10.1210/me.2005-0205.CrossRefGoogle Scholar
- 34.Zhou J, Liu M, Zhai Y, Xie W: The Anti-Apoptotic Role of Pregnane × Receptor in Human Colon Cancer Cells. Molecular endocrinology (Baltimore, Md). 2007.Google Scholar
- 36.Guevara I, Iwanejko J, Dembinska-Kiec A, Pankiewicz J, Wanat A, Anna P, Golabek I, Bartus S, Malczewska-Malec M, Szczudlik A: Determination of nitrite/nitrate in human biological material by the simple Griess reaction. Clin Chim Acta. 1998, 274 (2): 177-188. 10.1016/S0009-8981(98)00060-6.CrossRefPubMedGoogle Scholar
- 37.Bhalla S, Ozalp C, Fang S, Xiang L, Kemper JK: Ligand-activated pregnane × receptor interferes with HNF-4 signaling by targeting a common coactivator PGC-1alpha. Functional implications in hepatic cholesterol and glucose metabolism. The Journal of biological chemistry. 2004, 279 (43): 45139-45147. 10.1074/jbc.M405423200.CrossRefPubMedGoogle Scholar
- 39.Kliewer SA, Moore JT, Wade L, Staudinger JL, Watson MA, Jones SA, McKee DD, Oliver BB, Willson TM, Zetterstrom RH, et al: An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell. 1998, 92 (1): 73-82. 10.1016/S0092-8674(00)80900-9.CrossRefPubMedGoogle Scholar
- 40.Nagaoka R, Iwasaki T, Rokutanda N, Takeshita A, Koibuchi Y, Horiguchi J, Shimokawa N, Iino Y, Morishita Y, Koibuchi N: Tamoxifen activates CYP3A4 and MDR1 genes through steroid and xenobiotic receptor in breast cancer cells. Endocrine. 2006, 30 (3): 261-268. 10.1007/s12020-006-0003-6.CrossRefPubMedGoogle Scholar
- 42.Drocourt L, Pascussi JM, Assenat E, Fabre JM, Maurel P, Vilarem MJ: Calcium channel modulators of the dihydropyridine family are human pregnane × receptor activators and inducers of CYP3A, CYP2B, and CYP2C in human hepatocytes. Drug metabolism and disposition: the biological fate of chemicals. 2001, 29 (10): 1325-1331.Google Scholar
- 43.Cariou S, Donovan JC, Flanagan WM, Milic A, Bhattacharya N, Slingerland JM: Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America. 2000, 97 (16): 9042-9046. 10.1073/pnas.160016897.CrossRefPubMedPubMedCentralGoogle Scholar
- 44.Bouvard V, Zaitchouk T, Vacher M, Duthu A, Canivet M, Choisy-Rossi C, Nieruchalski M, May E: Tissue and cell-specific expression of the p53-target genes: bax, fas, mdm2 and waf1/p21, before and following ionising irradiation in mice. Oncogene. 2000, 19 (5): 649-660. 10.1038/sj.onc.1203366.CrossRefPubMedGoogle Scholar
- 56.Forrester K, Ambs S, Lupold SE, Kapust RB, Spillare EA, Weinberg WC, Felley-Bosco E, Wang XW, Geller DA, Tzeng E, et al: Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53. Proceedings of the National Academy of Sciences of the United States of America. 1996, 93 (6): 2442-2447. 10.1073/pnas.93.6.2442.CrossRefPubMedPubMedCentralGoogle Scholar
- 60.Binder C, Schulz M, Hiddemann W, Oellerich M: Induction of inducible nitric oxide synthase is an essential part of tumor necrosis factor-alpha-induced apoptosis in MCF-7 and other epithelial tumor cells. Laboratory investigation; a journal of technical methods and pathology. 1999, 79 (12): 1703-1712.PubMedGoogle Scholar
- 61.Galley HF, Nelson SJ, Dhillon J, Dubbels AM, Webster NR: Effect of the nitric oxide inhibitor, L-N(G)-monomethylarginine, on accumulation of interleukin-6 and interleukin-8, and nuclear factor-kappaB activity in a human endothelial cell line. Crit Care Med. 1999, 27 (5): 908-912. 10.1097/00003246-199905000-00024.CrossRefPubMedGoogle Scholar
- 62.Garvey EP, Oplinger JA, Furfine ES, Kiff RJ, Laszlo F, Whittle BJ, Knowles RG: 1400 W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo. The Journal of biological chemistry. 1997, 272 (8): 4959-4963. 10.1074/jbc.272.8.4959.CrossRefPubMedGoogle Scholar
- 67.Gu M, Lynch J, Brecher P: Nitric oxide increases p21(Waf1/Cip1) expression by a cGMP-dependent pathway that includes activation of extracellular signal-regulated kinase and p70(S6k). The Journal of biological chemistry. 2000, 275 (15): 11389-11396. 10.1074/jbc.275.15.11389.CrossRefPubMedGoogle Scholar
- 69.Meyer zu Schwabedissen HE, Tirona RG, Yip CS, Ho RH, Kim RB: Interplay between the nuclear receptor pregnane × receptor and the uptake transporter organic anion transporter polypeptide 1A2 selectively enhances estrogen effects in breast cancer. Cancer research. 2008, 68 (22): 9338-9347. 10.1158/0008-5472.CAN-08-0265.CrossRefPubMedPubMedCentralGoogle Scholar
- 71.Zhou C, Tabb MM, Sadatrafiei A, Grun F, Blumberg B: Tocotrienols activate the steroid and xenobiotic receptor, SXR, and selectively regulate expression of its target genes. Drug metabolism and disposition: the biological fate of chemicals. 2004, 32 (10): 1075-1082. 10.1124/dmd.104.000299.CrossRefGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/3/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.