![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
B Inhibition and Gene Expression Signature
Departments of Rheumatology (S.O., M.M., N.F.), Clinical Chemistry and Human Genetics (P.C., A.C., M.-P.M., V.B.), Hematology (E.C.), Pathology (L.d.L.), and Genetic Engineering (S.T.), Center for Biomedical Integrative Genoproteomics, University of Liège, Centre Hospitalier Universitaire Sart-Tilman, Liège, Belgium
Received October 31, 2005; accepted February 23, 2006
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
B (NF-B) activity in myeloma cells by removing p65 from its binding sites through estrogen receptor 
interaction with p65. It is noteworthy that microarray analysis showed that raloxifene treatment decreased the expression of known NF-B-regulated genes involved in myeloma cell survival and myeloma-induced bone lesions (e.g., c-myc, mip-1, hgf, pac1,...) and induced the expression of a subset of genes regulating cellular cycle (e.g., p21, gadd34, cyclin G2,...). In conclusion, raloxifene induces myeloma cell cycle arrest and apoptosis partly through NF-B-dependent mechanisms. These findings also provide a transcriptional profile of raloxifene treatment on multiple myeloma cells, offering the framework for future studies of selective estrogen receptor modulators therapy in multiple myeloma.
). Multiple myeloma treatment remains unsatisfactory, and new drugs targeting key signaling pathways required for myeloma growth or survival are needed.
Raloxifene is a selective estrogen receptor modulator (SERM) registered for the treatment of osteoporosis (Delmas et al., 2002). SERMs have both estrogen-agonistic and antagonistic properties depending on the tissue, the cell type, and even the target gene. Raloxifene thus has in vitro antiestrogen activities on breast tumor cells, and raloxifene treatment is associated with a decreased incidence of invasive breast cancer (Martino et al., 2004). However, raloxifene properties on the proliferation and survival of other cancer cells, including multiple myeloma cells, have not been explored extensively.
Many studies have reported the crucial role of the transcription factor NF-B in the growth and survival of various tumor cells (Panwalkar et al., 2004). The typical heterodimeric p50/p65 NF-B complex is sequestered in the cytoplasm through its association with IB family inhibitors. Activation by various stimuli leads to IB protein phosphorylation and degradation, allowing for NF-B translocation into the nucleus, where it specifically binds DNA and stimulates target gene transcription (Greten and Karin, 2004). In contrast to normal cells, NF-B is constitutively activated in various cancer cells, including myeloma cells. This constitutive activity favors cell proliferation, inhibits apoptosis, and enhances resistance to cytotoxic agents (Hideshima et al., 2002; Bharti et al., 2003). Moreover, elevated levels of NF-B activity were found in relapsing and refractory multiple myeloma (Feinman et al., 1999). Several established antimyeloma therapies, (e.g., dexamethasone, histone deacetylase inhibitors, and thalidomide) inhibit NF-B activity (Mitsiades et al., 2002b, 2004). It has been demonstrated that bortezomib (PS-341), a proteasome inhibitor, is highly efficient for the treatment of refractory multiple myeloma (Richardson et al., 2005), and it is believed that PS-341 efficacy against multiple myeloma occurs mainly as a result of the inhibition of NF-B activity (Mitsiades et al., 2002a).
The rationale for the investigation of raloxifene activity against multiple myeloma cells was based on the following scientific arguments: 1) some myeloma cells express estrogen receptors (ER) and 
(Otsuki et al., 2000); 2) tamoxifen has been shown to induce apoptosis in multiple myeloma cells and is currently tested in clinical trials (Treon et al., 1998; Fassas et al., 2001; Gauduchon et al., 2005); 3) ERs can inhibit NF-B activity in the presence of their ligands (Galien and Garcia, 1997; Harnish et al., 2000; Valentine et al., 2000); 4) raloxifene decreases osteoclastic bone resorption through modulation of cytokines such as IL-6 and the RANKL/OPG system (Cheung et al., 2003); and 5) raloxifene is a well tolerated drug largely used for the treatment of postmenopausal osteoporosis.
The aim of our study was thus to investigate the response of myeloma cells to raloxifene. We also characterized the signaling pathways involved in this effect and performed a microarray analysis to determine the transcriptional profile of raloxifene-treated multiple myeloma cells. These findings provide the framework for future studies of raloxifene activity in multiple myeloma.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Cells were grown in RPMI 1640 medium supplemented with 5 or 10% fetal calf serum, penicillin (100 IU/ml), and streptomycin (100 µg/ml). For all experiments, myeloma cells were plated in phenol red-free RPMI 1640 supplemented with 1% charcoal-treated fetal calf serum, penicillin (100 IU/ml), and streptomycin (100 µg/ml).
Raloxifene was a kind gift from Eli Lilly (Indianapolis, IN). Tamoxifen and 17-estradiol were purchased from Sigma-Aldrich (Schnelldorf, Germany). Raloxifene, tamoxifen, and 17-estradiol were dissolved in ethanol at a concentration of 1 mM. Vincristin sulfate was obtained from Teva Pharma (Wilrijk, Belgium). The broad-spectrum caspase inhibitor (Z-VAD-FMK) (Alexis Corporation, San Diego, CA) was dissolved in 10 mM DMSO. The NF-B inhibitor BAY 11-7085 and the proteasome inhibitor MG-132 (Alexis Corporation) were dissolved in 20 mM ethanol and in 20 mM DMSO, respectively.
MTS Reduction Assay. Myeloma cells were seeded at 2 x 104 cells/well in flat bottomed 96-well plates (VWR, Leuven, Belgium). Cell viability was assessed by reduction of the tetrazolium salt (MTS) to the formazan product in viable cells ("Cell Titer 96 Aqueous"; Promega, Leiden, The Netherlands) as described previously (Olivier et al., 2005). The data were expressed as a percentage of absorbance observed in untreated cells.
Cell Cycle Analysis. The JJN-3 and U266 cells were synchronized at S phase by incubation with an inhibitor of DNA synthesis, thymidine (2.5 mM), for 24 h. The cells were then washed and seeded at 106 cells/well in six-well plates. The cells were treated with raloxifene or tamoxifen for 16 or 24 h, washed twice in PBS, and fixed with 2 ml of ice-cold 70% ethanol before being centrifuged and incubated (at 37°C for 30 min) in PBS containing RNase (5 µg/ml) and propidium iodide (50 µg/ml). The cells were analyzed with a flow cytometer equipped with an argon laser emitting at 488 nm (Epics XL; Beckman Coulter Inc., Fullerton, CA). Red fluorescence intensity was measured using a 620 ± 15-nm band pass filter. Graphical and cell cycle analyses were performed with FlowJo version 6.01 software (Tree Star, Ashland, OR).
Western Blotting Assays. Total protein expression was estimated by Western blotting as described previously (Olivier et al., 2005). Anti-ER, anti-ER (sc-543 and sc-8974; Santa Cruz Biotechnology, Santa Cruz, CA), anti-PARP, anti-caspase 8, anti-caspase 9 (all from BD Biosciences, Erembodegem, Belgium), anti-Bid (sc-6538; Santa Cruz Biotechnology), anti-caspase 3 (Alexis Corporation), anti-Myc (sc-40; Santa Cruz Biotechnology), anti-p21WAF1 (Oncogene, Boston, MA), or anti-actin (Sigma-Aldrich) antibodies were used as primary antibodies. Horseradish peroxidase-linked anti-rabbit, anti-goat, or anti-mouse antibodies (DakoCytomation, Glostrup, Denmark) were used as secondary antibodies. The reaction was revealed with the enhanced chemiluminescence detection reagent (ECL kit; GE Healthcare, Buckinghamshire, UK).
Kinase Assay. For endogenous IKK activity, JJN-3 cells (5 x 106 cells) were treated with raloxifene for the indicated times and subsequently lysed with 1% Triton X-100 lysis buffer. Cellular extracts were incubated with 5 µl of anti-IKK
antibody (sc-8330) or 5 µl of the control anti-HA antibody (sc-805) (both from Santa Cruz Biotechnology) for 2 h at 4°C. Then, 100 µl of protein A-coupled Sepharose beads were added and incubated under gentle agitation overnight at 4°C. After three washes in lysis buffer, the kinase activity was assayed by using 1 µg of GST-IB as substrate.
Electrophoretic Mobility Shift Assay. Electrophoretic mobility shift assays were performed as described previously (Bureau et al., 2002). The B probe was 5'-TTGGCAACGGCAGGGGAATTCCCCTCTCCTTAGGTT-3'. Nuclear extract samples containing 5 µg of protein were mixed with 5 µl of binding buffer (20 mM HEPES, pH 7.6, 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 20% glycerol, 0.1% Nonidet P-40, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride), 2 µl of bovine serum albumin, 3 µl of polydIdC, and the 32P-labeled probe. The complexes were separated on a polyacrylamide gel. To confirm specificity, competition assays were performed with a 50-fold excess of unlabeled wild-type and nonspecific probes. For supershifting experiments, 1 µg of each antibody (p50, p65, RelB, and c-Rel, all from Santa Cruz Biotechnology, and p52 from Cell Signaling (Leiden, The Netherlands) was incubated with the extracts for 30 min on ice before the addition of the 32P-labeled probe.
Immunofluorescence. Myeloma cells (106 cells/well in six-well plates) were cytocentrifuged and fixed in 4% formaldehyde. The slides were blocked with PBS containing 1.5% dry milk before incubation with a goat anti-p65 antibody (1/100 dilution; Santa Cruz Biotechnology) for 45 min at 37°C. Slides were then washed in PBS and incubated with a secondary anti-goat antibody conjugated to fluorescein isothiocyanate (1/50 dilution; DakoCytomation) for 45 min at 37°C. Slides were washed in PBS and analyzed under a fluorescence microscope (magnification, 40x; Nikon Eclipse E800 with Sony DXC-9100P).
Chromatin Immunoprecipitation. Chromatin immunoprecipitation (ChIP) assays were performed with a ChIP assay kit (Upstate Biotechnology, Dundee, UK). Anti-p65 antibody (5 µl), 5 µl of antiestrogen receptor antibody, or 5 µl of the control anti-HA antibody (all from Santa Cruz Biotechnology) was used for the immunoprecipitation. The mip-1 promoter sequence was detected with the sense 5'-CTCTTCACACTCACAGGAGA-3' primer and the antisense 5'-TAGGCAGCCCTGGCGGAT-3' primer. PCR was performed using the AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA) according to the following protocol: 99°C for 3 min followed by 30 amplification cycles (95°C for 1 min, 50°C for 1 min, and 72°C for 1 min).
Immunoprecipitation. Nuclear and cytoplasmic protein extracts of JJN-3 cells (5 x 106 cells/dish) were prepared as described previously (Delhalle et al., 2002). For immunoprecipitation, nuclear extracts were incubated with 5 µl of anti-ER antibody or 5 µl of the control anti-HA antibody (sc-805) (both from Santa Cruz Biotechnology) for 2 h at 4°C. Then, 100 µl of protein A-coupled Sepharose beads were added and incubated under gentle agitation overnight at 4°C. After three washes in lysis buffer, bound proteins were resolved by SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting.
|
Real Time PCR. Real time PCR was carried out using the Taq-Man platform with the SYBR Green master mix (Applied Biosystems), as described previously (Heid et al., 1996). Primers, whose sequences were available upon request, were designed using the Primer Express software (Applied Biosystems).
Statistical Analysis. Data are presented as mean values ± S.E.M. (standard error of the mean). Data were analyzed by the Mann-Whitney test. In all analyses, p < 0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
and ER Expression in Multiple Myeloma Cell Lines. Before studying the effects of raloxifene on multiple myeloma cell growth and survival, we verified the expression of the two ER isoforms in JJN-3, U266, and RPMI 8226 cells. All cell lines expressed both ER and ER, as previously shown (Fig. 1A) (Treon et al., 1998).
|
-estradiol did not (Fig. 1B). Both raloxifene and tamoxifen also decreased U266 cell viability (Fig. 1B), but an equivalent cytotoxicity required a longer incubation in these cells. A weak effect of raloxifene and tamoxifen was also observed on RPMI 8226 cells after 72 h of treatment (cell viability was decreased by 23 and 17%, respectively) (data not shown).
The in vitro concentrations of raloxifene and tamoxifen required to induce apoptosis of breast cancer cells or glioma cells vary from 1 to 40 µM (Frasor et al., 2004; Hui et al., 2004). Based on these studies and on our dose-response data (not shown), all our experiments were carried out using a 5 µM concentration, which inhibited JJN-3 cell growth by 50%.
Next, we determined whether the exposure of multiple myeloma cells to these SERMs potentiates the cytotoxic effects of other chemotherapeutic drugs currently used in the treatment of multiple myeloma. Raloxifene or tamoxifen enhanced the cytotoxic effects of vincristine against JJN-3 and RPMI 8226 cells (Fig. 1C). After 72 h of treatment, vincristine (at 0.1 µg/ml), raloxifene, or tamoxifen (at 5 µM) alone decreased JJN-3 cell viability by 
50%, whereas the addition of raloxifene or tamoxifen to vincristine induced, respectively, a 76 and 82% decrease in JJN-3 cell viability (Fig. 1C). RPMI 8226 cells showed a similar response to vincristine alone, and both raloxifene and tamoxifen again enhanced vincristine-induced cell death (Fig. 1C). A synergistic effect between SERM and arsenic trioxide (at 5 µM) was also observed in RPMI 8226 myeloma cells (Fig. 1D), but such an effect could not be investigated in JJN-3 cells, because these cells are extremely sensitive to arsenic trioxide (data not shown). A weaker but reproducible additive cytotoxic effect was also observed when U266 cells were treated simultaneously with arsenic trioxide and SERMs (data not shown).
Fluorescence-activated cell sorting analysis showed that both raloxifene and tamoxifen (both at 5 µM) blocked the cell cycle before G2/M in JJN-3 cells, as shown by an increased proportion of cells in the S phase and a decrease of the number of cells in G2/M (Fig. 1E). In U266 cells, raloxifene and tamoxifen also blocked cell cycle progression but in the G2/M phase (data not shown).
Raloxifene and Tamoxifen Induce Myeloma Cell Apoptosis. To verify that raloxifene and tamoxifen induced cell death by apoptosis, we studied the activation of caspases and the cleavage of PARP in JJN-3 and U266 cells. Figure 2A shows that tamoxifen and raloxifene induced a proteolytic cleavage of caspase-8, caspase-3, and PARP (Fig. 2, A and B). The involvement of the mitochondrial apoptotic pathway was also shown by the cleavage of Bid and caspase-9 in JJN-3 cells after raloxifene and tamoxifen treatment (Fig. 2C). To confirm that raloxifene and tamoxifen induced caspase-dependent apoptosis, we tested the effect of the caspase inhibitor Z-VAD-FMK on cell death and PARP cleavage induced by raloxifene or tamoxifen. Pretreatment of JJN-3 cells with Z-VAD-FMK (at 50 µM) for 1 h before the addition of raloxifene or tamoxifen (both at 5 µM) had a cytoprotective effect on myeloma cells (Fig. 2D). In the same experimental conditions, the presence of the caspase inhibitor induced a marked decrease of raloxifene or tamoxifen-induced PARP cleavage in JJN-3 cells (Fig. 2E).
|
B Activity in Myeloma Cells. Because NF-B is constitutively active in multiple myeloma cells and promotes myeloma cell growth, survival, and drug resistance (Feinman et al., 1999; Bharti et al., 2003), we investigated the effects of the NF-B inhibitor BAY 11-7085 or the proteasome inhibitor MG-132 on JJN-3 cell viability. Increasing concentrations of BAY 11-7085 (1-4 µM) or MG-132 (0.1-0.4 µM) decreased JJN-3 viability after 24 h, as determined by the MTS reduction assay (Fig. 3A).
We next investigated whether the SERMs inhibited the IKK activity and NF-B-binding activity in myeloma cells. As shown by kinase assay performed with a GST-IB substrate, a marked IKK activity was observed in untreated JJN-3 cells, and raloxifene only weakly decreased this activity after 16 and 24 h of treatment (Fig. 3B). Consistent with these data, mobility shift assays also showed that NF-B was constitutively active in the multiple myeloma cell lines. Raloxifene decreased NF-B DNA-binding activity in JJN-3 or U266 cells (Fig. 3, C and D). In JJN-3 cells, the inhibition was already observed after 30 min and was complete after 16 h of treatment. A similar but less pronounced inhibition was observed after treatment of JJN-3 or U266 cells with the same concentration of tamoxifen (Fig. 3, C and D). NF-B DNA-binding complexes contained various homo- or heterodimers of p50, p52, p65, and RelB subunits in JJN-3 cells, whereas the NF-B complex was mainly constituted by p52/RelB subunits in U266 cells as demonstrated by supershift experiments (Fig. 3, C and D). Competition experiments performed with a 50-fold excess of an unlabeled palindromic b probe or a nonspecific probe confirmed the specificity of NF-B-binding activity in JJN-3 or U266 cells (Fig. 3, C and D).
To determine the subcellular localization of NF-B subunits, immunofluorescence staining was performed with an anti-p65 antibody. Although p65 was exclusively observed in the nucleus of untreated cells, raloxifene or tamoxifen treatment rapidly induced p65 relocalization to the cytoplasm of JJN-3 (Fig. 3E) and U266 cells (data not shown).
|
Interaction with p65. To study the mechanisms of raloxifene-mediated NF-B inhibition in myeloma cells, we investigated whether ER directly interacts with p65. Nuclear extracts from untreated or raloxifene-stimulated JJN-3 cells were immunoprecipitated with an anti-ER antibody, and the resulting precipitates were analyzed by an anti-p65 Western blotting analysis (Fig. 4A). Whereas p65 was weakly associated with ER in untreated cells, this interaction was strongly enhanced after 5 min of raloxifene treatment and became undetectable after longer stimulation (Fig. 4A). These data suggested that raloxifene may induce a conformation change in ER, resulting in decreased NF-B DNA-binding activity and, consequently, in the release and translocation of p65 from the nucleus to the cytoplasm.
To confirm this hypothesis, we evaluated the binding of p65 to NF-B-regulated promoters in the presence of raloxifene by ChIP assays. We selected the mip-1 gene promoter, because mip-1 is a known NF-B-regulated gene whose expression was significantly decreased after raloxifene treatment of JJN-3 cells (see Table 1 and Fig. 5A). Raloxifene treatment led to the dissociation of p65 (Fig. 4B, top left) and ER (Fig. 4B, middle left) binding to the mip-1 gene promoter. These data thus showed that p65 and ER are released from the NF-B-binding site on specific gene promoters upon treatment with raloxifene.
|
|
Gene Expression after Raloxifene Treatment. Gene expression in JJN-3 cells either untreated or treated with raloxifene for 2 h was compared by microarray analyses. This study identified 29 genes that were transiently repressed and 75 genes that were induced upon raloxifene treatment. A representative list of raloxifene target genes is shown in Table 1. It is noteworthy that 14 of these raloxifene-regulated genes are known to be associated with cell cycle and apoptosis signaling pathways (Table 1). Indeed, raloxifene down-regulated the transcription of the proto-oncogene c-myc and up-regulated the expression of the transcriptional repressor bcl-6 and of negative cell cycle regulators such as p21, cyclin G2, and e2f2 (Horne et al., 1997; Park et al., 2000; Shou et al., 2000; Zhu et al., 2001; Tang et al., 2002). Moreover, raloxifene suppressed the expression of other genes controlling multiple myeloma cell proliferation and/or survival including hgf and mip-1 (Anderson et al., 2002). Raloxifene also inhibited the expression of genes coding for the two mitogen-activated protein kinase phosphatases dusp2 (pac-1) and dusp4, two mitogen-induced early responsive genes overexpressed in breast cancer (Wang et al., 2003), and of the ras homolog arhe.
In addition, raloxifene up-regulated the expression of genes coding for the growth arrest and DNA damage-induced protein PP1R15A (Gadd34), the transcription factors ATF3 retinoic acid X receptor (rxrb), and CCAAT/enhancer-binding protein (C/EBP), the heat shock family protein 40 (DnaJB), and genes coding for the proteins involved in the response to endoplasmic reticulum stress (DnaJB, HER-PUD1) (Table 1) (Hasegawa et al., 2000; Fan et al., 2002). It also repressed the transcription factor 8 (tcf8) expression. Consistent with a recent study suggesting that tamoxifen-induced apoptotic response in glioma cells is mediated by an increase in cytosolic calcium (Hui et al., 2004), raloxifene up-regulated the expression of genes controlling intracellular calcium and chloride flux such as stc2, clic4, and clcn6 (Table 1).
To validate the microarray experiments, we confirmed by real time RT-PCR that raloxifene inhibited the expression of mip-1, c-myc, pac-1/dusp2, and less significantly, hgf, whereas it induced gadd 34, cyclin G2, and p21 gene expression in JJN-3 cells (Fig. 5A). Western blotting analyses also confirmed that raloxifene inhibited c-Myc and induced p21 expression in these cells (Fig. 5B). Likewise, in U266 cells, raloxifene up-regulated the expression of gadd 34, cyclin G2, and p21 genes and down-regulated c-myc but not mip-1 gene expression, whereas the expression of dusp2/pac-1 and hgf could not be detected by RT-PCR in these cells (Fig. 5A).
Because raloxifene inhibited constitutive NF-B activity, and because several of the raloxifene-regulated genes are known NF-B targets, JJN-3 cells were treated with the NF-B inhibitor BAY 11-7085 (20 and 40 µM) for 2 h. In these conditions, real time PCR showed a decrease in mip-1, c-myc, and hgf gene expression, thus indicating that the raloxifene-induced inhibition of these genes is most likely NF-B-mediated, whereas the drug did not influence pac1/dusp2 expression (Fig. 5C), indicating that raloxifene inhibited its expression through a pathway that is most probably NF-B-independent.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Martino et al., 2004). Indeed, multiple myeloma cells express ER, and previous studies demonstrated that tamoxifen or toremifene could induce multiple myeloma cell death and cell cycle arrest (Treon et al., 1998; Otsuki et al., 2000). We therefore studied the activity of raloxifene on ER-positive multiple myeloma cells, considering that the raloxifene antiresorptive effect on bones and its good clinical tolerance are additional arguments to justify such an investigation.
In our experimental conditions, we indeed observed that raloxifene blocked the cell cycle and induced apoptosis in several ER-positive multiple myeloma cell lines. Moreover, raloxifene also increased vincristine and arsenic trioxide cytotoxic response. To further characterize the mechanisms responsible for raloxifene in vitro activity, we first explored the NF-B pathway. Indeed, the NF-B transcription factor is constitutively active in refractory multiple myeloma, and several novel treatments, such as bortezomib, successfully target this factor (Hideshima et al., 2002; Richardson, 2003). Raloxifene could indeed inhibit NF-B activity, as demonstrated by the study of NF-B cellular localization, DNA binding, and transcriptional activities. Because ER has been demonstrated to interact with NF-B (Kalaitzidis and Gilmore, 2005), we further showed that raloxifene treatment led to a transiently increased ER/NF-B interaction, simultaneously with a loss of NF-B binding to specifically regulated gene promoters. These data suggest that raloxifene could indeed block the NF-B biological activity through a modulation of the ER association with p65, whereas raloxifene-induced inhibition of IKK activity was only partial.
We then performed a large scale study of gene expression regulation in multiple myeloma cells treated with raloxifene. This experiment led to the identification of only 104 genes whose expression is modified in response to this treatment. More significantly, only 29 genes showed reduced expression levels in response to raloxifene. However, many of the identified genes could provide us with a new insight into the mechanisms of raloxifene action, because 14 genes are known to regulate cell cycle or apoptosis, and several of them (c-myc or mip-1, for instance) have been previously identified as important regulators of multiple myeloma cell survival and disease progression. Mip-1 expression by tumor cells in the bone marrow of multiple myeloma patients correlates with an adverse prognosis (Terpos et al., 2003), and this gene is also a key regulator of bone resorption by osteoclasts and could therefore be responsible for the typical multiple myeloma bone lesions (Roodman and Choi, 2004).
It is very tempting to correlate the inhibition of NF-B with the list of genes obtained from the microarray experiment. Indeed, it is clear that some known NF-B-regulated genes, such as c-myc and mip-1, showed a decreased expression in response to raloxifene. We could also demonstrate that an NF-B inhibitor had a similar effect on them and that raloxifene treatment was associated with a loss of NF-B binding to the endogenous mip-1 promoter. However, as the number of down-regulated genes is quite low, it is likely that raloxifene inhibits only a subset of NF-B target genes. Indeed, although raloxifene blocked DNA-binding activities by several distinct NF-B complexes in the investigated cell lines, this inhibition is quite slow and incomplete. It is thus not surprising that, after 2 h of raloxifene treatment, only a part of the NF-B-regulated genes is affected. However, gene expression studies performed at later time points would identify many genes that are induced or repressed secondarily in the context of apoptosis or cell cycle arrest.
Raloxifene activity on multiple myeloma cells is clearly not restricted to the inhibition of the NF-B pathway. Indeed, the induction of cell cycle regulating genes (p21, cyclin G2) or ion channels as well as the inhibition of dusp phosphatase gene expression are likely to be NF-B-independent. Other specific antimyeloma therapeutic agents also combine NF-B-dependent and independent activities. For instance, the proteasome inhibitor bortezomib is highly efficient for the treatment of refractory multiple myeloma (Richardson et al., 2005). This drug blocks IB degradation and NF-B activation, but it also induces p53 and NOXA expression and increases the expression of cyclin-dependent kinase inhibitors including, as raloxifene, p21 (Mitsiades et al., 2002a; Hideshima et al., 2003; Qin et al., 2005).
In conclusion, raloxifene is a very promising drug for the treatment of multiple myeloma as it: 1) blocks cell cycle and induces apoptosis in multiple myeloma cells; 2) potentiates vincristine and arsenic trioxide cytotoxicity; 3) targets NF-B, a key regulator of multiple myeloma cell survival, associated with the progression of refractory multiple myeloma; 4) decreases IL-6 production by osteoblasts (Cheung et al., 2003); 5) inhibits osteoclast function and could thus limit bone resorption and favor the reparation of lytic lesions; and 6) is a well known and well tolerated compound. Given the poor prognosis of patients with refractory multiple myeloma and the current knowledge on raloxifene pharmacology, we believe that our data provide strong arguments for in vivo tests on animal models and a rapid onset of raloxifene clinical trials in these pathologies.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
V.B. and N.F. share equal responsibility in this work.
ABBREVIATIONS: SERM, selective estrogen receptor modulator; NF-B, nuclear factor-B; PS-341, bortezomib; ER, estrogen receptor; IL, interleukin; DMSO, dimethyl sulfoxide; BAY 11-7085, (E)3-[(4-t-butylphenyl)sulfonyl]-2-propenenitrile; MG-132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; PARP, poly(ADP-ribose) polymerase; IKK, IB kinase complex; HA, hemagglutinin; GST, glutathione S-transferase; PBS, phosphate-buffered saline; ChIP, chromatin immunoprecipitation; RT, reverse transcriptase; PCR, polymerase chain reaction; CT, control; RAL, raloxifene; TAM, tamoxifen; Z-VAD-FMK, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone.
Address correspondence to: Vincent Bours, Laboratory of Medical Chemistry and Human Genetics, CHU B35, Sart Tilman, 4000, Liège, Belgium. E-mail: vbours{at}ulg.ac.be
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bharti AC, Donato N, Singh S, and Aggarwal BB (2003) Curcumin (diferuloylmethane) down-regulates the constitutive activation of nuclear factor-B and IB kinase in human multiple myeloma cells, leading to suppression of proliferation and induction of apoptosis. Blood 101: 1053-1062.
Bureau F, Vanderplasschen A, Jaspar F, Minner F, Pastoret PP, Merville MP, Bours V, and Lekeux P (2002) Constitutive nuclear factor-kappaB activity preserves homeostasis of quiescent mature lymphocytes and granulocytes by controlling the expression of distinct Bcl-2 family proteins. Blood 99: 3683-3691.
Cheung J, Mak YT, Papaioannou S, Evans BA, Fogelman I, and Hampson G (2003) Interleukin-6 (IL-6), IL-1, receptor activator of nuclear factor B ligand (RANKL) and osteoprotegerin production by human osteoblastic cells: comparison of the effects of 17- oestradiol and raloxifene. J Endocrinol 177: 423-433.[Abstract]
Delhalle S, Deregowski V, Benoit V, Merville MP, and Bours V (2002) NF-kappaB-dependent MnSOD expression protects adenocarcinoma cells from TNF-alphainduced apoptosis. Oncogene 21: 3917-3924.[CrossRef][Medline]
Delmas PD, Ensrud KE, Adachi JD, Harper KD, Sarkar S, Gennari C, Reginster JY, Pols HA, Recker RR, Harris ST, et al. (2002) Efficacy of raloxifene on vertebral fracture risk reduction in postmenopausal women with osteoporosis: four-year results from a randomized clinical trial. J Clin Endocrinol Metab 87: 3609-3617.
Fan F, Jin S, Amundson SA, Tong T, Fan W, Zhao H, Zhu X, Mazzacurati L, Li X, Petrik KL, et al. (2002) ATF3 induction following DNA damage is regulated by distinct signaling pathways and over-expression of ATF3 protein suppresses cells growth. Oncogene 21: 7488-7496.[CrossRef][Medline]
Fassas AB, Rapoport AP, Bolanos-Meade J, Shanholtz C, Cottler-Fox M, and Tricot G (2001) Tamoxifen-based treatment induces clinically meaningful responses in multiple myeloma patients with relapsing disease after autotransplantation. Leuk Lymphoma 42: 1323-1328.[Medline]
Feinman R, Koury J, Thames M, Barlogie B, Epstein J, and Siegel DS (1999) Role of NF-kappaB in the rescue of multiple myeloma cells from glucocorticoid-induced apoptosis by bcl-2. Blood 93: 3044-3052.
Frasor J, Stossi F, Danes JM, Komm B, Lyttle CR, and Katzenellenbogen BS (2004) Selective estrogen receptor modulators: discrimination of agonistic versus antagonistic activities by gene expression profiling in breast cancer cells. Cancer Res 64: 1522-1533.
Galien R and Garcia T (1997) Estrogen receptor impairs interleukin-6 expression by preventing protein binding on the NF-kappaB site. Nucleic Acids Res 25: 2424-2429.
Gauduchon J, Gouilleux F, Maillard S, Marsaud V, Renoir JM, and Sola B (2005) 4-Hydroxytamoxifen inhibits proliferation of multiple myeloma cells in vitro through down-regulation of c-Myc, up-regulation of p27Kip1, and modulation of Bcl-2 family members. Clin Cancer Res 11: 2345-2354.
Greten FR and Karin M (2004) The IKK/NF-kappaB activation pathway—a target for prevention and treatment of cancer. Cancer Lett 206: 193-199.[CrossRef][Medline]
Harnish DC, Scicchitano MS, Adelman SJ, Lyttle CR, and Karathanasis SK (2000) The role of CBP in estrogen receptor cross-talk with nuclear factor-kappaB in HepG2 cells. Endocrinology 141: 3403-3411.
Hasegawa T, Xiao H, Hamajima F, and Isobe K (2000) Interaction between DNA-damage protein GADD34 and a new member of the Hsp40 family of heat shock proteins that is induced by a DNA-damaging reagent. Biochem J 352: 795-800.
Heid CA, Stevens J, Livak KJ, and Williams PM (1996) Real time quantitative PCR. Genome Res 6: 986-994.
Hideshima T, Chauhan D, Richardson P, Mitsiades C, Mitsiades N, Hayashi T, Munshi N, Dang L, Castro A, Palombella V, et al. (2002) NF-kappa B as a therapeutic target in multiple myeloma. J Biol Chem 277: 16639-16647.
Hideshima T, Mitsiades C, Akiyama M, Hayashi T, Chauhan D, Richardson P, Schlossman R, Podar K, Munshi NC, Mitsiades N, et al. (2003) Molecular mechanisms mediating antimyeloma activity of proteasome inhibitor PS-341. Blood 101: 1530-1534.
Horne MC, Donaldson KL, Goolsby GL, Tran D, Mulheisen M, Hell JW, and Wahl AF (1997) Cyclin G2 is up-regulated during growth inhibition and B cell antigen receptor-mediated cell cycle arrest. J Biol Chem 272: 12650-12661.
Hui AM, Zhang W, Chen W, Xi D, Purow B, Friedman GC, and Fine HA (2004) Agents with selective estrogen receptor (ER) modulator activity induce apoptosis in vitro and in vivo in ER-negative glioma cells. Cancer Res 64: 9115-9123.
Kalaitzidis D and Gilmore TD (2005) Transcription factor cross-talk: the estrogen receptor and NF-kappaB. Trends Endocrinol Metab 16: 46-52.[CrossRef][Medline]
Martino S, Cauley JA, Barrett-Connor E, Powles TJ, Mershon J, Disch D, Secrest RJ, and Cummings SR (2004) Continuing outcomes relevant to Evista: breast cancer incidence in postmenopausal osteoporotic women in a randomized trial of raloxifene. J Natl Cancer Inst 96: 1751-1761.
Mitsiades CS, Mitsiades NS, McMullan CJ, Poulaki V, Shringarpure R, Hideshima T, Akiyama M, Chauhan D, Munshi N, Gu X, et al. (2004) Transcriptional signature of histone deacetylase inhibition in multiple myeloma: biological and clinical implications. Proc Natl Acad Sci USA 101: 540-545.
Mitsiades N, Mitsiades CS, Poulaki V, Chauhan D, Fanourakis G, Gu X, Bailey C, Joseph M, Libermann TA, Treon SP, et al. (2002a) Molecular sequelae of proteasome inhibition in human multiple myeloma cells. Proc Natl Acad Sci USA 99: 14374-14379.
Mitsiades N, Mitsiades CS, Poulaki V, Chauhan D, Richardson PG, Hideshima T, Munshi N, Treon SP, and Anderson KC (2002b) Biologic sequelae of nuclear factor-kappaB blockade in multiple myeloma: therapeutic applications. Blood 99: 4079-4086.
Olivier S, Fillet M, Malaise M, Piette J, Bours V, Merville MP, and Franchimont N (2005) Sodium nitroprusside-induced osteoblast apoptosis is mediated by long chain ceramide and is decreased by raloxifene. Biochem Pharmacol 69: 891-901.[Medline]
Otsuki T, Yamada O, Kurebayashi J, Moriya T, Sakaguchi H, Kunisue H, Yata K, Uno M, Yawata Y, and Ueki A (2000) Estrogen receptors in human myeloma cells. Cancer Res 60: 1434-1441.
Panwalkar A, Verstovsek S, and Giles F (2004) Nuclear factor-kappaB modulation as a therapeutic approach in hematologic malignancies. Cancer 100: 1578-1589.[CrossRef][Medline]
Park WH, Seol JG, Kim ES, Hyun JM, Jung CW, Lee CC, Kim BK, and Lee YY (2000) Arsenic trioxide-mediated growth inhibition in MC/CAR myeloma cells via cell cycle arrest in association with induction of cyclin-dependent kinase inhibitor, p21, and apoptosis. Cancer Res 60: 3065-3071.
Qin JZ, Ziffra J, Stennett L, Bodner B, Bonish BK, Chaturvedi V, Bennett F, Pollock PM, Trent JM, Hendrix MJ, et al. (2005) Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells. Cancer Res 65: 6282-6293.
Richardson P (2003) Clinical update: proteasome inhibitors in hematologic malignancies. Cancer Treat Rev 29 (Suppl 1): 33-39.[CrossRef]
Richardson PG, Sonneveld P, Schuster MW, Irwin D, Stadtmauer EA, Facon T, Harousseau JL, Ben-Yehuda D, Lonial S, Goldschmidt H, et al. (2005) Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N Engl J Med 352: 2487-2498.
Roodman GD (2004) Pathogenesis of myeloma bone disease. Blood Cells Mol Dis 32: 290-292.[CrossRef][Medline]
Roodman GD and Choi SJ (2004) MIP-1 alpha and myeloma bone disease. Cancer Treat Res 118: 83-100.[Medline]
Shou Y, Martelli ML, Gabrea A, Qi Y, Brents LA, Roschke A, Dewald G, Kirsch IR, Bergsagel PL, and Kuehl WM (2000) Diverse karyotypic abnormalities of the c-myc locus associated with c-myc dysregulation and tumor progression in multiple myeloma. Proc Natl Acad Sci USA 97: 228-233.
Tang TT, Dowbenko D, Jackson A, Toney L, Lewin DA, Dent AL, and Lasky LA (2002) The forkhead transcription factor AFX activates apoptosis by induction of the BCL-6 transcriptional repressor. J Biol Chem 277: 14255-14265.
Terpos E, Politou M, Szydlo R, Goldman JM, Apperley JF, and Rahemtulla A (2003) Serum levels of macrophage inflammatory protein-1 alpha (MIP-1alpha) correlate with the extent of bone disease and survival in patients with multiple myeloma. Br J Haematol 123: 106-109.[CrossRef][Medline]
Treon SP, Teoh G, Urashima M, Ogata A, Chauhan D, Webb IJ, and Anderson KC (1998) Anti-estrogens induce apoptosis of multiple myeloma cells. Blood 92: 1749-1757.
Valentine JE, Kalkhoven E, White R, Hoare S, and Parker MG (2000) Mutations in the estrogen receptor ligand binding domain discriminate between hormone-dependent transactivation and transrepression. J Biol Chem 275: 25322-25329.
Wang HY, Cheng Z, and Malbon CC (2003) Overexpression of mitogen-activated protein kinase phosphatases MKP1, MKP2 in human breast cancer. Cancer Lett 191: 229-237.[CrossRef][Medline]
Zhu JW, Field SJ, Gore L, Thompson M, Yang H, Fujiwara Y, Cardiff RD, Greenberg M, Orkin SH, and DeGregori J (2001) E2F1 and E2F2 determine thresholds for antigen-induced T-cell proliferation and suppress tumorigenesis. Mol Cell Biol 21: 8547-8564.
This article has been cited by other articles:
|
|
 |
|
  J.-M. Renoir, C. Bouclier, A. Seguin, V. Marsaud, and B. Sola Antioestrogen-mediated cell cycle arrest and apoptosis induction in breast cancer and multiple myeloma cells J. Mol. Endocrinol., March 1, 2008; 40(3): 101 - 112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Shahjahan Miah, T. L. Hughes, and K. S. Campbell KIR2DL4 Differentially Signals Downstream Functions in Human NK Cells through Distinct Structural Modules J. Immunol., March 1, 2008; 180(5): 2922 - 2932. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. P. Tabruyn, C. Sabatel, N.-Q.-N. Nguyen, C. Verhaeghe, K. Castermans, L. Malvaux, A. W. Griffioen, J. A. Martial, and I. Struman The Angiostatic 16K Human Prolactin Overcomes Endothelial Cell Anergy and Promotes Leukocyte Infiltration via Nuclear Factor-{kappa}B Activation Mol. Endocrinol., June 1, 2007; 21(6): 1422 - 1429. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
