BACKGROUND
the wnt signaling pathway plays important roles in the regulation of cell proliferation, differentiation, and apoptosis  <cit> .
in the canonical wnt pathway, wnt initiates signaling events by binding to a receptor complex, consisting of a member of the frizzled  family, and the low-density lipoprotein-receptor-related proteins   <dig> or lrp <dig>  subsequently the cytoplasmic adaptor protein disheveled  is phosphorylated and inhibits glycogen synthase kinase -3β activity through its association with axin.
unphosphorylated β-catenin accumulates in the cytoplasm and translocates into the nucleus, where it interacts with members of t cell factor/lymphoid enhancer factor  family to activate transcription of wnt target genes  <cit> .
the β-catenin molecule is a key effector in the canonical wnt pathway.
however, not all wnt proteins activate the β-catenin complex.
some wnt family members, such as wnt <dig>  wnt5a and wnt <dig>  are able to initiate β-catenin-independent wnt signaling by binding to a frizzled receptor and possibly the coreceptor, knypek  or ror <dig> or ror <dig>  <cit> .
this leads to release of intracellular calcium and the activation of enzymes such as calcium/calmodulin dependent protein kinase ii  and protein kinase c , which exert antagonistic activity on the canonical wnt pathway.
calcium has been implicated as an important mediator of antagonism of canonical wnt signaling, acting at multiple points in the canonical wnt pathway  <cit> .
spiperone is a butyrophenone antipsychotic agent with dopamine and serotonin  receptor antagonist properties  <cit> .
it is also a high affinity ligand of sigma receptors  <cit> .
radiolabeled spiperone and its analogues have been widely used in assessing dopamine receptor function based on positron emission tomography  in humans.
in this study, we demonstrate that spiperone, but not other related psychotropic drugs blocks canonical wnt signaling activated by wnt and lrp <dig> by elevating intracellular calcium levels.
RESULTS
inhibition of canonical wnt signaling by spiperone
to identify antagonists of canonical wnt signaling, we used a cell-based topflash reporter system to screen the gen-plus drug library  that contains  <dig> fda-approved drugs.
in this system, transfected dvl  stimulated tcf/lef response elements in the topflash reporter gene.
inhibitors of wnt signaling were identified based on their ability to block the transcription of the reporter gene, but not a control gene.
small molecular compounds were screened at  <dig> μm and  <dig> μm.
the initial screen identified spiperone as an antagonist of wnt signaling.
to confirm the wnt inhibitory effect of spiperone, the topflash reporter was activated by wnt1/lrp <dig> or wnt3/lrp <dig>  dvl and β-catenin, respectively, in transient transfection assays.
treatment with  <dig> μm spiperone only weakly inhibited dvl- or β-catenin-mediated signaling , whereas a more potent effect was observed at higher concentration  .
surprisingly, treatment of the same dose of spiperone strongly blocked wnt signaling activated by wnt1/lrp <dig> and wnt3/lrp <dig>  respectively .
in control experiments, spiperone did not inhibit signals from reporter genes for nfat and activator protein  <dig>  .
these results suggest that spiperone may specifically inhibit wnt signaling by targeting the wnt/lrp complex.
spiperone was chosen for the further study because of its highly selective inhibitory effect on wnt/lrp-mediated signaling.
antipsychotic spiperone analogs have no inhibitory effect on wnt signaling
to determine whether spiperone-mediated inhibition of wnt signaling is associated with its antipsychotic effect, we examined other psychotropic drugs for their effects on wnt signaling.
these ligands include serotonin 5-ht <dig> receptor antagonists , 5-ht <dig> receptor agonist , 5-ht <dig> receptor antagonists , additional serotonin receptor antagonists  and a sigma receptor ligand   <cit> .
nan- <dig> and clozapine have been shown to be dopamine receptor antagonists, whereas uh- <dig> and way <dig> are dopamine receptor agonists  <cit> .
the topflash reporter was transfected into hek <dig> cells with expression plasmids for wnt <dig> and lrp <dig>  the cells were treated with increasing concentrations of the various psychotropic drugs.
surprisingly, none of the drugs tested except spiperone had the ability to block wnt1/lrp6-mediated signaling , suggesting that inhibition of wnt signaling is not related to compound interactions with dopamine, serotonin or sigma receptors.
spiperone displays a similar wnt inhibition profile to that of ionomycin and thapsigargin
a previous study demonstrated that ionomycin strongly inhibited wnt pathway activation induced by a wnt ligand, but had much less effect on β-catenin-mediated wnt signaling  <cit> .
the inhibition profile exerted by ionomycin treatment is similar to that of spiperone.
accordingly, we examined the wnt inhibitory effects of spiperone, ionomycin and thapsigargin.
ionomycin and thapsigargin are well known agents that mobilize intracellular calcium.
as shown in figure  <dig>  all three agents dramatically inhibited wnt signaling activated by wnt <dig> and lrp <dig>  in contrast, spiperone and thapsigargin weakly inhibited dvl-mediated signaling, whereas ionomycin had almost no inhibitory effect at the concentration tested .
we observed some inhibition in dvl-mediated transcription at higher drug concentrations, associated with cell toxicity .
these results suggest that spiperone, ionomycin and thapsigargin inhibit the wnt signaling pathway through a similar mechanism, that involves changes in intracellular calcium levels.
spiperone effects on calcium mobilization
in order to assess whether spiperone has the ability to elevate intracellular calcium levels, fluo- <dig> facs analysis was used.
as shown in figure 4a, spiperone and ionomycin increased intracellular calcium levels, whereas dtg had no affect under similar conditions.
in cells treated with ionomycin, calcium levels remained elevated throughout the recording period .
in contrast, calcium signals induced by spiperone were relatively transient, suggesting that the two drugs alter calcium concentrations by different mechanisms.
the effect of spiperone was dose-dependent, with a maximal increase in calcium levels occurring at  <dig> μm .
ionomycin, a calcium ionophore, increases calcium flux across membranes by shielding its polar electrical charge from the apolar lipid bilayer  <cit> .
thapsigargin is an inhibitor of the calcium atpase pump, which causes calcium release from the endoplasmic reticulum into the cytoplasm, leading to perturbations in calcium homeostasis  <cit> .
as expected, pretreatment with  <dig> μm thapsigargin could not abolish ionomycin-induced calcium release .
however, thapsigargin blocked the subsequent cellular calcium response to spiperone.
.
similarly, pretreatment with spiperone prevented the thapsigargin-induced calcium increase .
these results suggest that spiperone and thapsigargin may act by a similar mechanism to enhance intracellular calcium levels.
the pkc inhibitor gf109203x does not affect spiperone-mediated inhibition of wnt signaling
calcium is a ubiquitous second messenger used to regulate a wide range of cellular processes.
when intracellular calcium is increased, it interacts with protein kinase c , calmodulin, and with other intracellular molecules, to activate downstream signaling pathways.
to explore the role of pkc in spiperone-mediated inhibition of wnt signaling, a pkc inhibitor gf109203x was tested.
as indicated in figure  <dig>  gf109203x had no effect on spiperone-mediated inhibition of wnt signal transduction induced by wnt <dig> and lrp <dig>  while gf109203x alone slightly enhanced the dvl-initiated signaling pathway .
discussion
previous studies have demonstrated that noncanonical wnt family members repress canonical wnt signaling by inducing a calcium influx  <cit> .
some regulators of calcium signaling also have been shown to inhibit canonical wnt signaling  <cit> .
maye et al.
reported that an increase in intracellular calcium concentration induced by ionomycin led to inhibition of the canonical wnt pathway  <cit> .
they also noted that ionomycin strongly blocked wnt1-activated signaling, whereas its inhibition became much less effective with downstream pathway activators such as dvl and β-catenin  <cit> .
in this study, we identified spiperone as a calcium regulator.
like ionomycin and thapsigargin, spiperone specifically targets the wnt/lrp <dig> complex, and increases intracellular calcium levels.
however, it is unclear how calcium blocks wnt-activated signaling.
our results indicate that blocking pkc activity by gf109203x does not alter spiperone-mediated inhibition of wnt signaling, suggesting that the pkc activation pathway is not involved.
a recent study showed that ionomycin is a negative regulator of β-catenin/tcf signaling in colon cancer cells and its inhibitory mechanism is related to the decreased nuclear β-catenin products and to the suppressed binding of tcf complexes to consensus dna  <cit> .
however, this study did not check the effect of ionomycin on wnt signaling activated by wnt and lrp coreceptor.
in our experiment, we showed that spiperone and thapsigargin weakly inhibited dvl-mediated signaling .
we also noted that ionomycin elicited some inhibitory effect on dvl- and β-catenin-mediated signaling at concentrations equal to and above  <dig> μm .
therefore, in addition to its inhibition function on wnt/lrp complex, ionomycin may target multiple steps of the canonical wnt pathway.
spiperone is a psychotropic agent that acts as a potent dopamine d <dig>  serotonin 5-ht1a, and serotonin 5-ht2a antagonist  <cit> , and binds to sigma receptors with high affinity  <cit> .
spiperone also has been reported to have immunosuppressive effects in the mouse, and this action may be independent of its serotonin or dopamine receptor blocking properties  <cit> .
among the several related psychotropic drugs analyzed in the present study, only spiperone significantly blocked wnt signaling.
it will be interesting to determine whether spiperone-mediated wnt inhibition is associated with its immunosuppressive function, insofar as wnt-signaling is required for lymphocyte development.
during the preparation of this manuscript, liang et al.
<cit>  reported that spiperone stimulated calcium-dependent chloride secretion through a protein tyrosine kinase-coupled phospholipase c-dependent pathway, which supports our findings.
CONCLUSIONS
in summary, our result clearly demonstrated that spiperone is a calcium regulator.
it specifically blocks canonical wnt signaling by elevating intracellular calcium levels.
this drug may have chemopreventive or chemotherapeutic utility in malignancies associated with abnormal wnt activation.

 the wnt signaling pathway plays important roles in the regulation of cell proliferation, differentiation, and apoptosis. in the canonical wnt pathway, wnt initiates signaling events by binding to a receptor complex, consisting of a member of the frizzled family, and the low-density lipoprotein-receptor-related proteins or lrp subsequently the cytoplasmic adaptor protein disheveled is phosphorylated and inhibits glycogen synthase kinase -3β activity through its association with axin. unphosphorylated β-catenin accumulates in the cytoplasm and translocates into the nucleus, where it interacts with members of t cell factor/lymphoid enhancer factor family to activate transcription of wnt target genes. the β-catenin molecule is a key effector in the canonical wnt pathway. however, not all wnt proteins activate the β-catenin complex. some wnt family members, such as wnt wnt5a and wnt are able to initiate β-catenin-independent wnt signaling by binding to a frizzled receptor and possibly the coreceptor, knypek or ror or ror. this leads to release of intracellular calcium and the activation of enzymes such as calcium/calmodulin dependent protein kinase ii and protein kinase c, which exert antagonistic activity on the canonical wnt pathway. calcium has been implicated as an important mediator of antagonism of canonical wnt signaling, acting at multiple points in the canonical wnt pathway. spiperone is a butyrophenone antipsychotic agent with dopamine and serotonin receptor antagonist properties. it is also a high affinity ligand of sigma receptors. radiolabeled spiperone and its analogues have been widely used in assessing dopamine receptor function based on positron emission tomography in humans. in this study, we demonstrate that spiperone, but not other related psychotropic drugs blocks canonical wnt signaling activated by wnt and lrp by elevating intracellular calcium levels.  inhibition of canonical wnt signaling by spiperone to identify antagonists of canonical wnt signaling, we used a cell-based topflash reporter system to screen the gen-plus drug library that contains fda-approved drugs. in this system, transfected dvl stimulated tcf/lef response elements in the topflash reporter gene. inhibitors of wnt signaling were identified based on their ability to block the transcription of the reporter gene, but not a control gene. small molecular compounds were screened at μm and μm. the initial screen identified spiperone as an antagonist of wnt signaling. to confirm the wnt inhibitory effect of spiperone, the topflash reporter was activated by wnt1/lrp or wnt3/lrp dvl and β-catenin, respectively, in transient transfection assays. treatment with μm spiperone only weakly inhibited dvl- or β-catenin-mediated signaling, whereas a more potent effect was observed at higher concentration. surprisingly, treatment of the same dose of spiperone strongly blocked wnt signaling activated by wnt1/lrp and wnt3/lrp respectively. in control experiments, spiperone did not inhibit signals from reporter genes for nfat and activator protein. these results suggest that spiperone may specifically inhibit wnt signaling by targeting the wnt/lrp complex. spiperone was chosen for the further study because of its highly selective inhibitory effect on wnt/lrp-mediated signaling. antipsychotic spiperone analogs have no inhibitory effect on wnt signaling to determine whether spiperone-mediated inhibition of wnt signaling is associated with its antipsychotic effect, we examined other psychotropic drugs for their effects on wnt signaling. these ligands include serotonin 5-ht receptor antagonists, 5-ht receptor agonist, 5-ht receptor antagonists, additional serotonin receptor antagonists and a sigma receptor ligand. nan- and clozapine have been shown to be dopamine receptor antagonists, whereas uh- and way are dopamine receptor agonists. the topflash reporter was transfected into hek cells with expression plasmids for wnt and lrp the cells were treated with increasing concentrations of the various psychotropic drugs. surprisingly, none of the drugs tested except spiperone had the ability to block wnt1/lrp6-mediated signaling, suggesting that inhibition of wnt signaling is not related to compound interactions with dopamine, serotonin or sigma receptors. spiperone displays a similar wnt inhibition profile to that of ionomycin and thapsigargin a previous study demonstrated that ionomycin strongly inhibited wnt pathway activation induced by a wnt ligand, but had much less effect on β-catenin-mediated wnt signaling. the inhibition profile exerted by ionomycin treatment is similar to that of spiperone. accordingly, we examined the wnt inhibitory effects of spiperone, ionomycin and thapsigargin. ionomycin and thapsigargin are well known agents that mobilize intracellular calcium. as shown in figure all three agents dramatically inhibited wnt signaling activated by wnt and lrp in contrast, spiperone and thapsigargin weakly inhibited dvl-mediated signaling, whereas ionomycin had almost no inhibitory effect at the concentration tested. we observed some inhibition in dvl-mediated transcription at higher drug concentrations, associated with cell toxicity. these results suggest that spiperone, ionomycin and thapsigargin inhibit the wnt signaling pathway through a similar mechanism, that involves changes in intracellular calcium levels. spiperone effects on calcium mobilization in order to assess whether spiperone has the ability to elevate intracellular calcium levels, fluo- facs analysis was used. as shown in figure 4a, spiperone and ionomycin increased intracellular calcium levels, whereas dtg had no affect under similar conditions. in cells treated with ionomycin, calcium levels remained elevated throughout the recording period. in contrast, calcium signals induced by spiperone were relatively transient, suggesting that the two drugs alter calcium concentrations by different mechanisms. the effect of spiperone was dose-dependent, with a maximal increase in calcium levels occurring at μm. ionomycin, a calcium ionophore, increases calcium flux across membranes by shielding its polar electrical charge from the apolar lipid bilayer. thapsigargin is an inhibitor of the calcium atpase pump, which causes calcium release from the endoplasmic reticulum into the cytoplasm, leading to perturbations in calcium homeostasis. as expected, pretreatment with μm thapsigargin could not abolish ionomycin-induced calcium release. however, thapsigargin blocked the subsequent cellular calcium response to spiperone.. similarly, pretreatment with spiperone prevented the thapsigargin-induced calcium increase. these results suggest that spiperone and thapsigargin may act by a similar mechanism to enhance intracellular calcium levels. the pkc inhibitor gf109203x does not affect spiperone-mediated inhibition of wnt signaling calcium is a ubiquitous second messenger used to regulate a wide range of cellular processes. when intracellular calcium is increased, it interacts with protein kinase c, calmodulin, and with other intracellular molecules, to activate downstream signaling pathways. to explore the role of pkc in spiperone-mediated inhibition of wnt signaling, a pkc inhibitor gf109203x was tested. as indicated in figure gf109203x had no effect on spiperone-mediated inhibition of wnt signal transduction induced by wnt and lrp while gf109203x alone slightly enhanced the dvl-initiated signaling pathway. discussion previous studies have demonstrated that noncanonical wnt family members repress canonical wnt signaling by inducing a calcium influx. some regulators of calcium signaling also have been shown to inhibit canonical wnt signaling. maye et al. reported that an increase in intracellular calcium concentration induced by ionomycin led to inhibition of the canonical wnt pathway. they also noted that ionomycin strongly blocked wnt1-activated signaling, whereas its inhibition became much less effective with downstream pathway activators such as dvl and β-catenin. in this study, we identified spiperone as a calcium regulator. like ionomycin and thapsigargin, spiperone specifically targets the wnt/lrp complex, and increases intracellular calcium levels. however, it is unclear how calcium blocks wnt-activated signaling. our results indicate that blocking pkc activity by gf109203x does not alter spiperone-mediated inhibition of wnt signaling, suggesting that the pkc activation pathway is not involved. a recent study showed that ionomycin is a negative regulator of β-catenin/tcf signaling in colon cancer cells and its inhibitory mechanism is related to the decreased nuclear β-catenin products and to the suppressed binding of tcf complexes to consensus dna. however, this study did not check the effect of ionomycin on wnt signaling activated by wnt and lrp coreceptor. in our experiment, we showed that spiperone and thapsigargin weakly inhibited dvl-mediated signaling. we also noted that ionomycin elicited some inhibitory effect on dvl- and β-catenin-mediated signaling at concentrations equal to and above μm. therefore, in addition to its inhibition function on wnt/lrp complex, ionomycin may target multiple steps of the canonical wnt pathway. spiperone is a psychotropic agent that acts as a potent dopamine d serotonin 5-ht1a, and serotonin 5-ht2a antagonist, and binds to sigma receptors with high affinity. spiperone also has been reported to have immunosuppressive effects in the mouse, and this action may be independent of its serotonin or dopamine receptor blocking properties. among the several related psychotropic drugs analyzed in the present study, only spiperone significantly blocked wnt signaling. it will be interesting to determine whether spiperone-mediated wnt inhibition is associated with its immunosuppressive function, insofar as wnt-signaling is required for lymphocyte development. during the preparation of this manuscript, liang et al. reported that spiperone stimulated calcium-dependent chloride secretion through a protein tyrosine kinase-coupled phospholipase c-dependent pathway, which supports our findings.  in summary, our result clearly demonstrated that spiperone is a calcium regulator. it specifically blocks canonical wnt signaling by elevating intracellular calcium levels. this drug may have chemopreventive or chemotherapeutic utility in malignancies associated with abnormal wnt activation. 
wnt signaling affects fundamental development pathways by regulating cell proliferation and differentiation. instead, spiperone increases intracellular calcium levels in a similar manner to thapsigargin, that also impedes wnt signal transduction. it inhibits wnt signaling by enhancing intracellular calcium levels. inhibition of protein kinase c had no effect on spiperone-mediated antagonism of wnt signaling. we identified spiperone, a psychotropic drug, as a novel wnt inhibitor, which specifically blocks canonical wnt signaling prior to the activation of β-catenin.
 wnt signaling affects fundamental development pathways by regulating cell proliferation and differentiation. aberrant activation of wnt/β-catenin signaling promotes the development of several cancers and is an attractive target for chemopreventive and chemotherapeutic agents.  in order to identify the novel antagonists for the wnt/β-catenin pathway, we employed a cell-based wnt reporter system to screen a library of known drugs. we identified spiperone, a psychotropic drug, as a novel wnt inhibitor, which specifically blocks canonical wnt signaling prior to the activation of β-catenin. the wnt inhibitory function of spiperone is not associated with its dopamine-, serotonin- and sigma-receptor antagonist properties. instead, spiperone increases intracellular calcium levels in a similar manner to thapsigargin, that also impedes wnt signal transduction. inhibition of protein kinase c had no effect on spiperone-mediated antagonism of wnt signaling.  spiperone is a calcium regulator. it inhibits wnt signaling by enhancing intracellular calcium levels. 