Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Aug 9;108(32):13253-7.
doi: 10.1073/pnas.1110431108. Epub 2011 Jul 25.

Salinomycin inhibits Wnt signaling and selectively induces apoptosis in chronic lymphocytic leukemia cells

Desheng Lu  1 , Michael Y Choi, Jian Yu, Januario E Castro, Thomas J Kipps, Dennis A Carson
Affiliations

Salinomycin inhibits Wnt signaling and selectively induces apoptosis in chronic lymphocytic leukemia cells

Desheng Lu et al. Proc Natl Acad Sci U S A. .

Abstract

Salinomycin, an antibiotic potassium ionophore, has been reported recently to act as a selective breast cancer stem cell inhibitor, but the biochemical basis for its anticancer effects is not clear. The Wnt/β-catenin signal transduction pathway plays a central role in stem cell development, and its aberrant activation can cause cancer. In this study, we identified salinomycin as a potent inhibitor of the Wnt signaling cascade. In Wnt-transfected HEK293 cells, salinomycin blocked the phosphorylation of the Wnt coreceptor lipoprotein receptor related protein 6 (LRP6) and induced its degradation. Nigericin, another potassium ionophore with activity against cancer stem cells, exerted similar effects. In otherwise unmanipulated chronic lymphocytic leukemia cells with constitutive Wnt activation nanomolar concentrations of salinomycin down-regulated the expression of Wnt target genes such as LEF1, cyclin D1, and fibronectin, depressed LRP6 levels, and limited cell survival. Normal human peripheral blood lymphocytes resisted salinomycin toxicity. These results indicate that ionic changes induced by salinomycin and related drugs inhibit proximal Wnt signaling by interfering with LPR6 phosphorylation, and thus impair the survival of cells that depend on Wnt signaling at the plasma membrane.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Salinomycin specifically inhibits the Wnt/β-catenin signaling pathway initiated by Wnt1. (A) HEK293 cells were transfected with a SuperTOPflash reporter and empty vector or Wnt1 expression plasmid. (B) An AP1-luciferase reporter was transfected with a vector expressing constitutively active Ha-Ras (RasV12) into HEK293 cells. (C) HEK293 cells were transfected with an NFAT-luciferase reporter along with NFATc expression plasmid. The transfected cells were treated with salinomycin at increasing concentrations (from 4 nM to 1,250 nM) for 24 h. The luciferase activities were measured and values were normalized to β-gal activity. Results are expressed as fold induction compared with empty vector control. (D) HEK293 cells were transfected with empty vector or Wnt1 expression plasmid. Then cells were treated with the indicated amounts of salinomycin for 16 h. The β-catenin and β-actin levels were detected by Western blot analysis.
Fig. 2.
Fig. 2.
Salinomycin inhibits Wnt/β-catenin signaling via at least two different mechanisms. The SuperTOPflash reporter was transfected into HEK293 cells along with expression plasmids encoding Wnt1 (A), Fzd5 and LRP6 (B), and β-catenin (C), respectively. After transfection for 24 h, cells were treated with indicated amounts of salinomycin for another 24 h. Luciferase activity was measured and presented as fold induction compared with empty vector control. (D) The inhibitory effects of salinomycin on Wnt/β-catenin signaling mediated by Wnt1 or Fzd5/LRP6 or β-catenin were compared and presented as relative inhibition rate (as percentage).
Fig. 3.
Fig. 3.
Salinomycin selectively targets the Wnt/Fzd/LRP complex. (A) Salinomycin selectively inhibited Wnt signaling mediated by Wnt1/Fzd5/LRP6 or Wnt3/Fzd5/LRP6 or DKK2/Fzd5/LRP6. The SuperTOPflash reporter assays were performed on HEK293 cells transfected with the indicated expression vectors. The cells were treated with either vehicle (DMSO) or salinomycin (250 nM and 500 nM) for a further 24 h. The cells were harvested and luciferase activity was measured as described in Materials and Methods. (B) The relative inhibition rate (as percentage) was used to compare the inhibitory effects of salinomycin on Wnt signaling induced by Fzd5/LRP6 or Wnt1/Fzd5/LRP6 or Wnt3/Fzd5/LRP6 or DKK2/Fzd5/LRP6. (C) HEK293 cells were transfected with empty vector and Wnt1 expression vector for 24 h. Then cells were treated with the indicated amounts of salinomycin or vehicle control for 16 h. The phospho-LRP6 (Ser1490) and total LRP6 were detected by Western blot analysis. β-Actin was used as a loading control.
Fig. 4.
Fig. 4.
Comparison of the Wnt inhibitory effects of salinomycin, nigericin, and thapsigargin. HEK293 cells were cotransfected with the SuperTOPflash reporter construct, along with expression plasmids for Wnt1 (A), Fzd5/LRP6 (B), and β-catenin (C), respectively. After transfection for 24 h, the cells were treated with salinomycin (250 nM and 500 nM), thapsigargin (10 nM and 50 nM), and nigericin (125 nM, 250 nM, and 500 nM) as indicated. Luciferase activity is presented as fold induction compared with empty vector control.
Fig. 5.
Fig. 5.
Salinomycin selectively induced apoptosis in CLL cells. Primary CLL cells from 13 patients and the PBMCs from seven normal donors were treated with increasing amounts of salinomycin for 48 h. Apoptosis was measured by flow cytometry by using DiOC6/PI staining.
Fig. 6.
Fig. 6.
Salinomycin reduced the LRP6 protein level and down-regulated the expression of LEF1, cyclin D1, and fibronectin in CLL cells. (A) Primary CLL cells from two patients were treated with indicated amounts of salinomycin for 16 h. The phospho-LRP6 (Ser1490), total LRP6, and β-actin were detected by Western blot. (B) Primary CLL cells from four patients were treated with increasing concentrations of salinomycin for 16 h. The mRNA levels of LEF1, cyclin D1, and fibronectin were measured by real-time PCR as described in Materials and Methods.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Clevers H. Wnt/beta-catenin signaling in development and disease. Cell. 2006;127:469–480. - PubMed
    1. Moon RT, Kohn AD, De Ferrari GV, Kaykas A. WNT and beta-catenin signalling: Diseases and therapies. Nat Rev Genet. 2004;5:691–701. - PubMed
    1. Nusse R. Wnt signaling in disease and in development. Cell Res. 2005;15:28–32. - PubMed
    1. Willert K, Jones KA. Wnt signaling: is the party in the nucleus? Genes Dev. 2006;20:1394–1404. - PubMed
    1. Mao B, et al. LDL-receptor-related protein 6 is a receptor for Dickkopf proteins. Nature. 2001;411:321–325. - PubMed

Publication types

MeSH terms