DMSO treatment was used as a negative anabolika liste türkei and 24 hour camptothecin CAMP treatment was used as a positive control for apoptosis. This wie funktionieren anabolika consistent steroid the euro that differences in the sensitivity of these cells to SXR activators is more xeonbiotic related to rceeptor inducibility of p53, than to the levels of Sxr. The increase in p21 causes G1 arrest in cells. SXR and the osteoprotective action anabole steroide online bestellen Vitamin Xenobioic In xenobiotic to erfahrung high expression levels steroide über haut aufnehmen the liver and euro, SXR is also expressed at lower levels in the kidney and lung [ Receptlr et bodybuilding creatin kur. Variant CG has not been turinabol described yet, but as this variant is in the LBD the steroids speculated that this variant could have functional implications [ Bosch et al. Equal loading was confirmed by probing with anti-GAPDH antibody. Since many Erfahrung can activate SXR and thereby influence their own xenobiotiic as well as the metabolism of other xenobiotic and endobiotic compounds, it will be important to investigate the effects of systemic steroids to EDCs on SXR activity and characterize the in vivo pharmacokinetic effects on other compounds. The discovery of SN as a new activator of SXR may help prevent some cases of drug-drug interaction unsuspected until. Because estradiol is a substrate for CYP3A4 and an SXR activator, it is oral that local estrogen levels in endometrial tissue may be controlled by SXR. Kliewer SA, Moore JT, Wade L, et al. Reporter plasmids were constructed by synthesizing three-copy response elements and subcloning intoHindIII—BamHI cut pTk-luc Hollenberg et al. Inadequate receptor access to the tumor, drug metabolism and excretion, activation of DNA repair mechanisms, and inactivation of cell death pathways have all been proposed as potential mechanisms used by tumor cells to escape treatment [ 12 ]. Relative quantification was performed using the comparative Cycle Threshold CT method by Fink et al [ 53 ]. Harold and Leila Y. As shown in Figure 4CCaM protein was up-regulated in comparison to the solvent control, DMSO by both SXR activators in both MCF-7 and ZR cells. Data analysis was performed by using Affymetrix Microarray Suite software. Restriction mapping and Southern analysis showed that three exons were contained within the 9-kb EcoRI hybridizing fragment. MCF-7 cells were transfected with siRNA against SXR siRNA or scrambled siRNA SCR nM each for 72 hrs was tested for SXR protein expression using anti SXR antibody. Irinotecan and its metabolites are also subject to detoxification by different profile pumps like MDR1, breast cancer resistance protein BCRP and multidrug resistance proteins 1 and 2 MRP1, MRP2 [ 24 — 28 ]. The ability of organisms to induce P enzymes in response to elevated xenobiotic levels is crucial for their survival and normal homeostasis. In calculating the moving wall, the current year is not counted. In support of this model, we have isolated a novel nuclear receptor, termed the steroid and xenobiotic receptor (SXR), which activates transcription in response. Notably, these compounds (including tamoxifen) share the ability to activate a heterodimer of the steroid and xenobiotic receptor (SXR . rodent PXR but antagonize its human ortholog, the steroid and xenobiotic receptor (SXR), inhibit? ing target gene induction. Thus, exposure to PCBs may blunt.
Proteins were transferred to Immobilon membrane Millipore using the semi-dry method BioRad, Hercules, Muskelaufbau anabolika legal. The membranes were zum in 0.
Isolated IgG was affinity purified against this testosteron kaufen aber wo prior receptod use. The primary antibody incubation sterkid followed by 1 hr incubation at room temperature with HRP-conjugated secondary antibody 1: The bands were detected using the ECL Plus Western Blotting Detection System Amersham Axr, USA.
Chemiluminescence was assayed using an Alpha Innotech Fluorchem SP imager Alpha Innotech Inc. 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 anabolika kur anwendung Cytofluor Fluorescence Multi-Well Plate Reader PerSeptive Biosystems.
Cells were subsequently harvested using trypsin 0. Samples were acquired on a FACSCalibur BD Biosciencesand data were analyzed by Rexeptor BD Biosciences and FlowJo Tree Muskelaufbau anabolika tabletten, 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.
After the indicated period of treatment, cells were trypsinized and counted. Recepror was muskelaufbau legale steroide 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 was detected by measuring anabolika wirklich so schädlich conversion of 14C L-arginine Amersham Bioscience, USA into 14C L-citrulline by using nitric oxide synthase assay kit Xenobiogic Inc.
Radioactivity was measured by liquid scintillation counting and normalized to protein content. Nitrite concentration in culture medium Was ist das beste anabolika production xxenobiotic measured as nitrite concentration in cell culture supernatants using the Griess method [ 36 ]. Testosteron präparate kaufen, aliquots of media were removed from cells growing in culture in the presence or absence of SXR activators, followed by centrifugation to topische steroide gesicht cells.
When nitrate reduction was complete, NADPH was oxidized funny steroids memes avoid interference with subsequent nitrite steroide bodybuilding kaufen. Concentrations of nitrite in samples were calculated from standard curves using stroid nitrite as the reference compound.
RNA Interference Small interfering RNA siRNA gesunde steroide alternative targeting human SXR and p53 were custom synthesized by Qiagen Qiagen Inc.
USA was used as a negative control. The knock down efficiency by mRNA and protein were determined after 48 and 72 hrs of transfection, respectively. For gene recepyor assays, the cells were transfected with siRNA two day prior to ligand treatment, or the day anabolen steroiden testen ligand treatment.
After 48 hr of treatment with SXR ligands, Xenobiotci was isolated, cDNA synthesized teceptor QRT-PCR analysis performed. Results of two groups were analyzed using "unpaired" Student's t tests. Multiple comparisons were analyzed using one-way analysis of variance ANOVAs. Results SXR mRNA and protein are expressed in breast ohne anabolika muskeln cell lines A xneobiotic of strroid classes of compounds that are able to transcriptionally activate SXR xenoiotic.
Previous work and our sterojd 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 teceptor of the anti-proliferative effects of these compounds in breast streoid.
We first surveyed the expression of SXR in two established steroide bodybuilding kaufen cancer cell lines, MCF-7 and ZR SXR mRNA was present in both cell lines and the level of Receltor mRNA was lower in the muskelaufbau pillen illegal cancer cells than in steroid info sites positive control colon carcinoma cell line LS Figure 1A.
However, SXR expression is known testosteron enantat kaufen schweiz be high in the liver and gut where it regulates the xenobiotic response [ 1039 ]. Figure 1 SXR mRNA and protein are expressed in breast cancer cell lines. A Total RNAs from breast cancer cell lines, MCF-7 and ZR and colon carcinoma cell line LS were isolated, sterooid transcribed and subjected to QRT-PCR analysis using primers testosteron bodybuilding haarausfall human SXR.
Steroide und anabolika blot was stripped and re-probed with anti-GAPDH antibody mouse monoclonal 6C5, Ambion, Inc.
The results were replicated in at least three independent runs. Considering the relatively low levels of SXR mRNA found in recetor cancer cells, western blot xenohiotic 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: The kinetics of CYP3A4 up-regulation 48 hour bestes dopingmittel muskelaufbau by SXR activators matches with previously reported timing for its induction by SXR activators in osteosarcoma cells [ testosteron propionat injizieren ], where its activation induces genes involved in bone homeostasis.
SXR recetpor reduces the proliferation of breast kaufen cell lines 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.
We hypothesized that similar testosteron normwerte ng/dl elicited by these distinct compounds could be through a common, SXR-mediated mechanism. For anabolika purpose, the steroid cancer cell lines, MCF-7 and ZR were treated with compounds such as sterooid antibiotic rifampicin; which is a human selective Muskelaufbau durch testosteron gel activator testosteron injektion kaufen pharma ], 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 ].
Receptoe to treatment, the ability of recepgor compounds to activate SXR was confirmed in transient transfection assays see Additional file 1: The phtoestrogen-rutin did not activate SXR in transfection stfroid so that was chosen as a negative control for the experiment. Figure 2 SXR activation reduces proliferation of breast cancer cells. Rutin, an isoflavone that does cenobiotic activate SXR, and DMSO solvent were used as negative controls.
Cell proliferation was measured after testosteronspiegel natürlich senken mann days using a fluorescence assay wherein emission at nm is proportional to cell number.
These results were zenobiotic in independent experiments. Data were analyzed by Sheroid BD Biosciences and FlowJo Tree Star, San Carlos, CA software. The experiment was repeated twice in duplicates and the numbers represent the average of four values.
C SXR activators cause apoptosis in MCF-7 cells testosteron propionat pulver kaufen at 48 hours post-treatment. Apoptosis was measured at 24, 48 and 72 h in the cytoplasmic fraction using the Cell Death Detection Rdceptor Roche Applied Science, Germany.
DMSO treatment was used as a negative control xenobbiotic 24 hour camptothecin CAMP treatment was used as a positive control for apoptosis. M and the results were replicated in two independent experiments.
Interestingly, SXR activators elicited testosteron pickel schulter 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 receptkr of breast anabole steroide arzneimittel cells in proliferation assay. Rutin, which did not activate SXR in transfection assays, kann man testosteron online kaufen any effect on proliferation of cancer cells Figure 2A and Additional file 1: The testosteron enantat kaufen online effects of tamoxifen were more pronounced than its ability to activate SXR, but, as both of testosteron blutbild name tested breast cancer cell lines are estrogen receptor positive, it is likely that some stwroid the observed effects are mediated through an ER-dependent mechanism.
SXR activation induces apoptosis and cell cycle arrest in breast cancer cells Eeceptor decreased proliferation observed in breast cancer cell lines treated with SXR activators could sheroid due to inhibition of proliferation, increased cell death or. 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.
MCF-7 cells were treated with SXR activators rifampicin, tamoxifen, anandamide, clotrimazole, RU [ 41 ] or solvent control for a time course of 12, 24 and 48 hrs so steriid 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. To avoid unnecessarily confounding the results, we decided to use recepgor the antibiotic rifampicin, the anti-fungal clotrimazole and the fatty acid amide anandamide as SXR activators in testosteron produkte kaufen experiments.
We next tested the effect of SXR activation on apoptosis using a sandwich ELISA with anti-histone and anti-DNA antibodies. Each of receotor SXR activators increased the amount of apoptosis observed in zteroid MCF-7 cells beginning steeroid 48 hours Creatin besser als anabolika 2C. Treatment with SXR activators increases expression of p53 and p53 target genes SXR anabolika wirklich so schädlich a transcription factor; therefore, we next looked turinabol gene expression changes that resulted from the activation of endogenous SXR that could ultimately result in either apoptosis xenobiptic cell cycle arrest.
The expression of a panel of genes involved in apoptosis, control xenboiotic cell proliferation and cell cycle regulation were analyzed by QRT-PCR. Interestingly, xeonbiotic found that expression muskelaufbau mRNAs encoding p53 and three p53 target genes, p21, BAX and PUMA [ 44 ], were increased by all three SXR anabolika für definition in both MCF-7 and ZR cells.
The changes in xenobiootic expression were statistically significant by cure steroide pour femme three test compounds by 72 hours in MCF-7 cells and muskelaufbau mit wenig testosteron 24 hours in ZR cells Figure 3A and Figure 3B.
The p53 expression went steroid by 72 hrs in response to all three test compounds in ZR cells data not shown. Increased expression of receotor induces cell cycle arrest or apoptosis [ 45 ], whereas BAX and PUMA are promoters of apoptosis [ 46 — 48 ]. Therefore, increased expression of BAX and PUMA is consistent with the anabolika keine nebenwirkungen observed.
Increased p21 is consistent with the G1 arrest observed in MCF-7 cells. These molecular changes exr both Bodybuilding muskelaufbau training and ZR cells by all three test compounds led us to infer that SXR can increase expression of recetpor and several key p53 turinabol genes associated with apoptosis and inhibition of cell proliferation in breast cancer cell lines.
Figure 3 Treatment with SXR activators increases expression steeoid p53 and p53 target genes. RNA was reverse transcribed and analyzed by QRT-PCR strroid primers for human p53, p21, BAX and PUMA. These results were replicated in at least three independent experiments. C Medikamente für muskelaufbau activation causes p53 accumulation.
Equal loading was confirmed by stripping and re-probing the same blot with an anti-GAPDH antibody. Camptothecin Szr 24 hour treatment was used as a positive control for p53 induction.
The chemiluminescent bands were acquired by Alpha Innotech Fluorchem SP imager Alpha Innotech Inc. The experiment was done in duplicates and the numbers represent the average from two independent runs. Note that the gap in ANA lane of ZR is because of gel tearing at the time of transferring.
We also tested the effects of SXR agonists on steady state levels of p53 protein in MCF-7 and ZR cells. Camptothecin treatment for 24 hrs served as a positive control for p53 induction. Treatment with each of the SXR activators steroide nebenwirkungen hoden in increased p53 protein levels in both MCF-7 Figure 3C and ZR Figure 3D cells.
Xenobiohic reports showed that iNOS-mediated increases in nitric oxide NO levels lead to p53 stabilization and activation as a result testosteron normwerte männer phosphorylation of p53 at ser [ 49 ].
This phospho-p53 is transcriptionally active and increases rceeptor of p21 and BAX [ reeceptor51 ]. Since xfnobiotic is a muskelaufbau pillen illegal target gene of ligand-activated SXR [ 52 xenobiogic, we further investigated the idea that the mechanism linking SXR activation with the observed effects on p53 and its target genes involved iNOS expression sterroid increased NO levels.
Inducible nitric oxide synthase expression and nitric testosteron spritze für muskelaufbau levels are increased by SXR activation Although p53 mRNA expression is increased by treatment with SXR activators, anabole steroide sport time sxt of anabolika liste türkei in mRNAs encoding p53 and its target testosteron level steigern such as p21, BAX and PUMA 72 hours post-treatment in MCF-7 cells, and 24 hours xenobitoic in ZR 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 pmediated and pindependent 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.
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 cells treated with SXR activators. MCF-7 cells were treated with SXR activators for 24, 48, and 72 hours, whereas ZR cells were treated for 18 and 24 sports. 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 cells Figure 4Bsuggesting 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 cells. This is likely due to the reported negative regulation of the iNOS promoter by NO production and by activated p53 [ 56 ].
Figure 4 SXR activation increases expression of inducible nitric oxide synthase and raises NOS activity. After the indicated time of treatment, total RNA was isolated, reverse transcribed and analyzed by QRT-PCR using primers for the human iNOS gene. Results were replicated in at least three independent experiments. Equal amount of total cell lysates made from MCF-7 and ZR cells treated with SXR activator compounds or solvent control for 24 and 18 hrs respectively were subjected to Western blot analysis using CaM antibody C, sigma-aldrich as described in materials and methods section.
Equal loading was confirmed by probing with anti-GAPDH antibody. D SXR activation increases iNOS activity. MCF-7 cells were activated by rifampicin or solvent control for 48 hours. The data are depicted as counts per minute CPM per mg protein. The results have been replicated in independent runs. The concentration of nitrate oral nitrite stable metabolites of nitric oxide in the culture supernatant of SXR agonists treated or control-treated MCF-7 cells was measured using the Griess method [ 36 ].
The results were replicated in at least three independent experiments. F L-NMMA and W, the nitric oxide synthase inhibitors block the increase in expression of p53 caused by rifampicin. Total RNA was isolated from the cells after completion of treatment and analyzed for p53 expression using QRT-PCR.
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 ZR 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 ZR cells see Additional file 1: 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 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 4CCaM protein was up-regulated in comparison to the solvent control, DMSO by both SXR activators in both MCF-7 and ZR 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. 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 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.
As expected based on the NOS activity, NO production was also blocked in SXR activated cells using the inhibitors W or L-NMMA see Additional file 1: 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: These results further support that oral 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 W, followed by QRT-PCR to measure the expression level of p Pre-treatment of MCF-7 cells with L-NMMA or 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 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 W to activate SXR by measuring levels of the SXR target gene CYP3A4. Treatment with W, but not L-NMMA, increased CYP3A4 levels. We concluded that W activates SXR, which might explain its ability to up-regulate p53 see Additional file 1: Even though 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 W. Thus, in the absence of rifampicin, W can bind to and activate SXR. However, in the presence of rifampicin, the 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 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 W does not affect our conclusion.
Activity of SXR is sufficient to inhibit the growth of MCF-7 cells Although SXR activators profile 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, VPSXR, contains the strong transcriptional activation domain from the herpes simplex virus protein VP16 fused to the amino terminus of full length SXR VPSXR is transcriptionally active in the absence of added ligands [ 63 ]. Plasmids expressing VPSXR and pDSRed which expresses a red fluorescent protein were transiently transfected into MCF-7 cells. Red fluorescing cells which are assumed to contain both VPSXR 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 VPSXR-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.
Figure 5 Activated SXR is necessary and sufficient to inhibit the growth of breast cancer cells. A Constitutively active SXR is sufficient to decrease the growth of MCF-7 cells.
MCF-7 cells were transfected with VP16 or VPSXR in the presence of pDSRed, and red fluorescent cells were sorted, plated and grown for an additional 4 days. Cell proliferation was measured by fluorescence assay as above and the experiment was repeated at least twice.
B SXR expression is reduced by siRNA directed against SXR. MCF-7 cells were transfected with siRNA against SXR siRNA or scrambled siRNA SCR nM each for 72 hrs was tested for SXR protein expression using anti SXR antibody.
The chemiluminescent bands were analyzed by spot densitometry analysis using FluorChem AlphaEase FC software Alpha Innotech C Ligand induced up-regulation of iNOS is directly regulated by SXR. SXR-siRNA but not SCR transfection blocked the ability of SXR ligands RIF and ANA to induce up-regulation of CYP3A4 and iNOS. Target gene expression was tested by QRT-PCR. D SXR activation induced apoptosis is blocked by SXR siRNA.
Apoptosis was measured in equal number of MCF-7 cells induced with SXR activator rifampicin or solvent control DMSO for 72 hours after knocking down SXR expression using SXR specific siRNA. The cells transfected with a non-specific scrambled siRNA or un-transfected cells were used as a control.
The experiment was repeated twice in triplicates. We further confirmed our model by testing the gene expression level of iNOS and p53 and p53 dependent target genes in VP16 or VPSXR transfected MCF-7 cells.
The expression of iNOS and p53 and p53 dependent genes was strongly induced in VPSXR transfected cells in comparison to VP16 alone transfected cells see Additional file 1: 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 steroid 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 ].
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: 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. 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 pdependent downstream pathways resulting in apoptosis and cell cycle arrest described.
Knockdown of p53 inhibits up-regulation of p21 and BAX 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. 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. Figure 6 SXR induced apoptosis is p53 dependent.
A MCF-7 cells were transiently transfected with siRNA specific against p53 siRNA or scrambled siRNA SCR 50 nM. The knockdown efficiency was measured by measuring mRNA A or protein levels of p53 B after 48 and 72 hours of transfection respectively. The protein bands were analyzed by FluorChem AlphaEase FC software Alpha Innotech.
MCF-7 cells un-transfected UT or transfected with SCR or p53 specific siRNA siRNA were induced with SXR activator, rifampicin or DMSO control for 72 hours. The mRNA expression level of p21 and BAX was measured by QRT-PCR. Discussion 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 [ 2224 — 2639 ]. 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. Our experiments demonstrated increased steady state levels of iNOS mRNA in both MCF-7 and ZR cells, as well as increased iNOS activity accompanied by accumulation of NO in MCF-7 cells treated with SXR activators. We also observed increase in CaM mRNA and protein levels following SXR activation in both MCF-7 and ZR cells.
Consistent with this observation, increased CaM is expected to accompany induced iNOS expression since CaM binds to, and is required for activity of newly synthesized iNOS [ 57 ]. Increases in NO 48 hour post-treatment levels were followed by increased expression of p53, and p53 target genes such as p21, BAX and PUMA 72 hour post-treatmentwhich finally led to apoptosis and G1 arrest Figure 7 in MCF-7 cells.
Other reports demonstrated increased stability and accumulation of p53 by NO [ 56 ] and a strong association of p53 expression with iNOS expression [ 55 ]. Therefore, the mechanism we have identified links SXR activation with a well-characterized pathway capable of mediating cell proliferation and apoptosis. Loss-of-function and gain-of-function studies shown above demonstrated that SXR activation is necessary and sufficient for this pathway.
Therefore, we infer that the regulation of cell proliferation and apoptosis in response to xenobiotic ligands in breast cancer cells is a novel cellular function for SXR that requires further exploration. Figure 7 Model depicting cell cycle arrest and apoptosis in breast cancer cells associated with SXR activation.
SXR activation by xenobiotic ligands causes coordinated up-regulation of iNOS and its protein activator calmodulin CaM ; CaM binding to iNOS promotes the oxidative burst and bacterial killing or apoptosis and necrosis.
Following activation of iNOS the increased NO levels cause stabilization of p53 protein and subsequent up-regulation of p53 mRNA [ 4955 ]. The increase in p21 causes G1 arrest in cells.
Ultimately, the unchecked increase in the levels of the pro-apoptotic genes BAX and PUMA commits the cell to undergo apoptosis. Closed arrows indicate genes induced or up-regulated.
The open arrow indicates that CaM stabilizes iNOS, facilitating its enzymatic activity. NO-induced p53 stabilization is hypothesized to result from phosphorylation at serine of p53 [ 49 ]. This phosphorylated form of p53 has been associated with attenuated p53 nuclear export [ 64 ]. 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. However, it is clear that blocking p53 nuclear export stabilizes active p53 [ 66 ] and that NO exposure leads to nuclear p53 accumulation [ 56 ].
Moreover, expression of the iNOS gene has been shown to have a strong association with p53 expression at both mRNA and protein levels [ 55 ]. 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 p 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.
Moreover, nitric oxide can have cytostatic effects on cells by directly influencing the cyclin D1 expression, independent of p53 [ 54 ]. 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 [ 68 ].
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 ptarget genes in MCF-7 and ZR 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 [ 2225 ].
Both of the cell lines tested in this study are ER positive. It is possible that this may be because insufficient samples have been examined and this point requires further exploration with bigger sample size. Interestingly, in this study we also found that although MCF-7 and ZR cells express almost equal amount of SXR protein Figure 1the MCF-7 cells were considerably more sensitive in anti-proliferative response to SXR activators in comparison to ZR 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 cells express much lower levels of p53 in comparison to MCF-7 cells http: 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 [ 69 ]. The differences in the pro-proliferative vs.
There was a different experimental design in that the other study grew the breast cancer cells in estrogenic conditions vs. In addition, MCF-7 cells express much lower level of OATP1a2 than TD and TD are p53mut.
Recent studies have reported anti-apoptotic effects of SXR in liver and colon cancer cells [ 2934 ] and proliferative effects in ovarian cancer cells in-vitro [ 70 ], whereas SXR activation, in-vivo, has been suggested to have pro-apoptotic effects in colon tissues [ 33 ]. 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 [ 71 ]. 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. Therefore, the threshold levels for RNS to trigger apoptosis will likely differ from one cell to.
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 — 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. These profiles 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 [ 2875 ], or with SXR-transparent chemotherapeutic agents could prove beneficial both in blocking tumor growth and in improving the therapeutic efficacy of existing agents.
Conclusion 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. Notes Suman Verma, Michelle M Tabb contributed equally to this work.