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.

Cell lines were treated with SXR ligands for the indicated times followed by isolation of total cellular RNA using Trizol reagent (Invitrogen). After removal of potentially contaminating genomic DNA by DNAse digestion and LiCl precipitation, 1 μg total RNA was reverse transcribed using Superscript III (Invitrogen) following the manufacturer's recommended protocol. Quantitative real time RT-PCR (QRT-PCR) was performed using primer sets as shown in Table 1 using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) or FastStart SYBR Green Master Mix (Roche Applied Science, Indianapolis, USA) in a DNA Engine Opticon-Continuous Fluorescence Detection System (MJ Research, Reno, NV). All samples were quantitated by the comparative cycle threshold C(t) method for relative quantitation of gene expression, normalized to either GAPDH or β-actin 35.

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.

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.

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.

NO production was measured as nitrite concentration in cell culture supernatants using the Griess method 36. 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 36. 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' 2737 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.

A variety of different classes of compounds that are able to transcriptionally activate SXR (e.g., anandamide, rexinoids, tamoxifen) have been previously observed to slow the proliferation of breast cancer cell lines and/or slow tumor growth in rodent models of breast cancer 34738. Previous work and our preliminary results showed that SXR was expressed in human breast cancers and in some breast cancer cell lines 2325. Therefore, we hypothesized that SXR activation might mediate some of the anti-proliferative effects of these compounds in breast cancer. We first surveyed the expression of SXR in two established breast cancer cell lines, MCF-7 and ZR-75-1. SXR mRNA was present in both cell lines and the level of SXR mRNA was lower in the breast cancer cells than in the positive control colon carcinoma cell line LS180 (Figure 1A). However, SXR expression is known to be high in the liver and gut where it regulates the xenobiotic response 1039.

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 24, where its activation induces genes involved in bone homeostasis.

Next, to test our hypothesis that SXR might play a role in some of the anti-proliferative effects previously observed by different classes of compounds (e.g., anandamide, rexinoids, tamoxifen); we did an initial screening experiment in which we tested a large number of structurally and functionally diverse compounds that all share the ability to activate SXR. We hypothesized that similar effects elicited by these distinct compounds could be through a common, SXR-mediated mechanism. For this purpose, the breast cancer cell lines, MCF-7 and ZR-75-1 were treated with compounds such as macrolide antibiotic rifampicin; which is a human selective SXR activator 10, the anti-estrogen tamoxifen; which is also an SXR agonist 1440, the fatty acid ethanolamide anandamide, the anti-fungal clotrimazole 41 or the calcium channel blocker nifedipine 42. Prior to treatment, the ability of these compounds to activate SXR was confirmed in transient transfection assays (see Additional file 1: supplementary Figure 2A). The phtoestrogen-rutin did not activate SXR in transfection assays so that was chosen as a negative control for the experiment.

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.

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 41 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 43. 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.

SXR is a transcription factor; therefore, we next looked for gene expression changes that resulted from the activation of endogenous SXR that could ultimately result in either apoptosis or cell cycle arrest. MCF-7 and ZR-75-1 cells were treated with 10 μM rifampicin, anandamide, clotrimazole or solvent controls, and total RNA was prepared after 24, 48 and 72 hours of incubation. The expression of a panel of genes involved in apoptosis, control of cell proliferation and cell cycle regulation were analyzed by QRT-PCR. Interestingly, we found that expression of mRNAs encoding p53 and three p53 target genes, p21, BAX and PUMA 44, were increased by all three SXR activators in both MCF-7 and ZR-75-1 cells. The changes in gene expression were statistically significant by all three test compounds by 72 hours in MCF-7 cells and by 24 hours in ZR-75-1 cells (Figure 3A and Figure 3B). The p53 expression went down by 72 hrs in response to all three test compounds in ZR-75-1 cells (data not shown). Increased expression of p21 induces cell cycle arrest or apoptosis 45, whereas BAX and PUMA are promoters of apoptosis 464748. Therefore, increased expression of BAX and PUMA is consistent with the apoptosis observed. Increased p21 is consistent with the G1 arrest observed in MCF-7 cells. These molecular changes in both MCF-7 and ZR-75-1 cells by all three test compounds led us to infer that SXR can increase expression of p53 and several key p53 target genes associated with apoptosis and inhibition of cell proliferation in breast cancer cell lines.

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 49. This phospho-p53 is transcriptionally active and increases expression of p21 and BAX 5051. Since iNOS is a reported target gene of ligand-activated SXR 52, 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.

Although p53 mRNA expression is increased by treatment with SXR activators, the time course of induction in mRNAs encoding p53 and its target genes such as p21, BAX and PUMA (72 hours post-treatment in MCF-7 cells, and 24 hours post-treatment in ZR-75-1 cells) led us to hypothesize that elevation of p53 mRNA levels and the resulting changes in p53 target gene expression were a secondary response to an earlier primary effect of SXR activation. The p53 tumor suppressor can be activated or stabilized in response to DNA damage or cellular stress such as accumulation of reactive oxygen species (ROS) or reactive nitrogen species (RNS) 53. Moreover, RNS-stabilized p53 has been shown to up-regulate both p21 and BAX expression 5051. Nitric oxide itself increases expression of p21 and causes late G1 arrest in cells through p53-mediated and p53-independent pathways 5054. Therefore, we considered the possibility that treatment with the various SXR activators was increasing the expression of a cellular stressor such as RNS. A previous report suggested that the promoter of inducible nitric oxide synthase (iNOS/NOS2A) contained an SXR-responsive element, and that increased expression of iNOS is a primary and direct response to SXR activation 52. Production of iNOS increases the cellular levels of NO and RNS and a significant association between iNOS and p53 expression has been demonstrated at the mRNA and protein levels 55. Accordingly, we examined expression of iNOS mRNA in MCF-7 and ZR-75-1 cells treated with SXR activators. MCF-7 cells were treated with SXR activators for 24, 48, and 72 hours, whereas ZR-75-1 cells were treated for 18 and 24 hours. These time points were selected because they precede those at which we observed up-regulation of p53 and its target genes in these cells, thus allowing the detection of prior events. We found that three different SXR activators increased iNOS mRNA levels in MCF-7 (Figure 4A) and ZR-75-1 cells (Figure 4B), suggesting that SXR activation, per se, and not other properties of the compounds was increasing iNOS mRNA levels. Increased iNOS expression was observed as early as 24 hours post-treatment in MCF-7 cells, whereas it could be detected starting at 18 hrs in ZR-75-1 cells. iNOS expression subsequently decreased in clotrimazole treated MCF-7 cells at 48 and 72 hours and in ZR-75-1 cells at 24 hrs. This is likely due to the reported negative regulation of the iNOS promoter by NO production and by activated p53 56.

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 57. 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 58. 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 59. 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 36. 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 60. 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) 61 or the iNOS specific inhibitor 1400 W 62 (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.

Although SXR activators were able to inhibit breast cancer cell proliferation, the compounds tested can potentially act through other pathways to stop cancer cell growth. To establish whether SXR activation, per se, was responsible for the observed effects on cell proliferation, we employed a constitutively active form of SXR 63. This construct, VP16-SXR, contains the strong transcriptional activation domain from the herpes simplex virus protein VP16 fused to the amino terminus of full length SXR-1. VP16-SXR is transcriptionally active in the absence of added ligands 63. Plasmids expressing VP16-SXR and pDSRed (which expresses a red fluorescent protein) were transiently transfected into MCF-7 cells. Red fluorescing cells (which are assumed to contain both VP16-SXR and pDSRed) were separated from non-fluorescing cells using fluorescence-activated cell sorting. The sorted red fluorescent cells were cultured for an additional four days, then harvested for cell proliferation assays. Proliferation of VP16-SXR-transfected cells was strongly inhibited compared with cells transfected with empty plasmid, or with VP16 alone (Figure 5A). Therefore, we infer that SXR activation itself decreased the proliferation of breast cancer cells, supporting our model that the test compounds also exerted their effects on proliferation through SXR.

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.

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 2737. 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.

To further confirm that activation of SXR and its subsequent anti-proliferative effects in breast cancer cells are mediated through a p53 dependent mechanism, we knocked down p53 using siRNA. Transient transfection of MCF-7 cells with p53 specific siRNA (siRNA) but not with a scrambled siRNA (SCR) reduced p53 mRNA level by more than 90% (Figure 6A) and camptothecin stabilized p53 protein level by more than 70% (Figure 6B). As predicted, induction of p21 and BAX mRNA by rifampicin is significantly blocked in p53 siRNA transfected, but not in SCR siRNA transfected cells (Figure 6C). These results confirm the requirement of p53 for the SXR-mediated growth inhibitory and apoptotic effects on breast cancer cells.