Spiperone enhances intracellular calcium level and inhibits the Wnt signaling pathway

Abstract

Background: Wnt signaling affects fundamental development pathways by regulating cell proliferation and differentiation. Aberrant activation of Wnt/beta-catenin signaling promotes the development of several cancers and is an attractive target for chemopreventive and chemotherapeutic agents.

Results: In order to identify the novel antagonists for the Wnt/beta-catenin pathway, we employed a cell-based Wnt reporter system (TOPflash) to screen a library of 960 known drugs. We identified spiperone, a psychotropic drug, as a novel Wnt inhibitor, which specifically blocks canonical Wnt signaling prior to the activation of beta-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.

Conclusion: Spiperone is a calcium regulator. It inhibits Wnt signaling by enhancing intracellular calcium levels.

Figure 1

Inhibition of Wnt signaling by spiperone. HEK293 cells were cotransfected with a TOPflash reporter construct, along with vectors for: (A) control (pcDNA3 plasmid alone), Wnt1/LRP6, or Wnt3/LRP6; (B) control (pcDNA3 plasmid alone) or Dvl; (C) control (pcDNA3 plasmid alone) or β-catenin. (D) HEK293 cells were transfected with an NFAT-Luc reporter and an expression plasmid for NFATc. (E) HEK293 cells were transfected with an AP1-Luc reporter and an expression plasmid for H-Ras^V12. After transfection for 24 h, the cells were treated with or without spiperone (5 μM) for another 24 h, and then harvested, and extracted for determination of luciferase activities. The β-galactosidase control plasmid was used to correct for transfection efficiency. The results are expressed as fold induction of luciferase activity normalized to a β-galactosidase control, and are the means of three experiments ± SEM.

Figure 2

Effect of psychotropic drugs on Wnt signaling activated by Wnt1 and LRP6. The TOPflash reporter was transfected into HEK293 cells with expression plasmids encoding Wnt1 and LRP6. After transfection, cells were treated with increasing concentrations of antipsychotic spiperone analogs for 24 h, as indicated. Then cells were harvested and luciferase activities were determined.

Figure 3

Spiperone, ionomycin and thapsigargin inhibit Wnt1- and LRP6-initiated Wnt signaling. HEK293 cells were transfected with a TOPflash reporter and expression plasmids for Wnt1, LRP6 and Dvl as indicated in the figure. After transfection for 24 h, cells were grown with 5 μM spiperone, 2 μM ionomycin, 50 nM thapsigargin and vehicle control for another 24 h. Then luciferase values were determined.

Figure 4

Spiperone induce a rise in intracellular calcium levels. (A) Tracing of fluo-4 fluorescence in cells stimulated with 10 μM spiperone, 10 μM DTG and 2 μM ionomycin over time. Drugs were added at 30 sec. (B) Tracing of fluo-4 fluorescence in cells stimulated with increasing doses of spiperone. Drugs were added at 30 sec. (C) Pretreatment with 1 μM thapsigargin could not abolish ionomycin-induced calcium release. Drugs were added at 10 and 225 sec as indicated by the arrowheads. (D) Thapsigargin blocks the subsequent cellular calcium responses to spiperone. Intracellular Ca²⁺ was recorded after stimulation with 1 μM thapsigargin and 10 μM spiperone. Drugs were added at 30 and 225 sec as indicated by the arrowheads. (E) Spiperone prevented thapsigargin-induced calcium increase. Intracellular calcium was traced after stimulation with 10 μM spiperone and 1 μM thapsigargin. Drugs were added at 30 and 225 sec as indicated by the arrowheads.

Figure 5

The protein kinase C inhibitor GF109203X does not affect spiperone-mediated inhibition of Wnt signaling. A TOPflash reporter was transfected into HEK293 cells with expression plasmids for Wnt1, LRP6 and Dvl as indicated in figure. After transfection, cells were treated with 5 μM spiperone, 5 μM GF109203X and combined use of two drugs for another 24 h. Then luciferase values were determined.