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Cited by 231Wnt/β-catenin signaling in development and cancerHedgehog Signaling Pathway StudiesDrug Transport and Resistance Mechanisms

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Niclosamide Suppresses Cancer Cell Growth By Inducing Wnt Co-Receptor LRP6 Degradation and Inhibiting the Wnt/β-Catenin Pathway

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Lu, Wenyan, Lin, Cuihong, Roberts, Michael J., Waud, William R., Piazza, Gary A., Li, Yonghe·2011·PLoS ONE
DOI 10.1371/journal.pone.0029290PMC PMC3241710
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Abstract

The Wnt/β-catenin signaling pathway is important for tumor initiation and progression. The low density lipoprotein receptor-related protein-6 (LRP6) is an essential Wnt co-receptor for Wnt/β-catenin signaling and represents a promising anticancer target. Recently, the antihelminthic drug, niclosamide was found to inhibit Wnt/β-catenin signaling, although the mechanism was not well defined. We found that niclosamide was able to suppress LRP6 expression and phosphorylation, block Wnt3A-induced β-catenin accumulation, and inhibit Wnt/β-catenin signaling in HEK293 cells. Furthermore, the inhibitory effects of niclosamide on LRP6 expression/phosphorylation and Wnt/β-catenin signaling were conformed in human prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. Moreover, we showed that the mechanism by which niclosamide suppressed LRP6 resulted from increased degradation as evident by a shorter half-life. Finally, we demonstrated that niclosamide was able to induce cancer cell apoptosis, and displayed excellent anticancer activity with IC(50) values less than 1 µM for prostate PC-3 and DU145 and breast MDA-MB-231 and T-47D cancer cells. The IC(50) values are comparable to those shown to suppress the activities of Wnt/β-catenin signaling in prostate and breast cancer cells. Our data indicate that niclosamide is a unique small molecule Wnt/β-catenin signaling inhibitor targeting the Wnt co-receptor LRP6 on the cell surface, and that niclosamide has a potential to be developed a novel chemopreventive or therapeutic agent for human prostate and breast cancer.

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Figure 1 Effects of niclosamide on Wnt3A and LRP6-induced Wnt/β signaling in HEK293 cells. (A) HEK293 cells in 6-well plates were treated with Wnt3A CM (20%) and niclosamide at indicated concentrations for 24 h. The levels of cytosolic free β-catenin, total cellular β-catenin, LRP6 and phospho-LRP6 (p-LRP6) were examined. All the samples were also probed with anti-actin antibody to verify equal loading. (B) HEK293 cells in 24-well plates were transiently transfected with LRP6 plasmid or the corresponding control vector, along with TOPFlash construct and β-galactosidase-expressing vector in each well. After being incubated for 24 h, cells were treated with niclosamide or niclosamide plus Wnt3A CM at indicated concentrations for 24 h. The luciferase activity was then measured 24 h later with normalization to the activity of the β-galactosidase. Values are averages of three determinations with the standard deviations indicated by error bars. **P<0.01 compared to the control cells without niclosamide treatment.
Figure 1 Effects of niclosamide on Wnt3A and LRP6-induced Wnt/β signaling in HEK293 cells. (A) HEK293 cells in 6-well plates were treated with Wnt3A CM (20%) and niclosamide at indicated concentrations for 24 h. The levels of cytosolic free β-catenin, total cellular β-catenin, LRP6 and phospho-LRP6 (p-LRP6) were examined. All the samples were also probed with anti-actin antibody to verify equal loading. (B) HEK293 cells in 24-well plates were transiently transfected with LRP6 plasmid or the corresponding control vector, along with TOPFlash construct and β-galactosidase-expressing vector in each well. After being incubated for 24 h, cells were treated with niclosamide or niclosamide plus Wnt3A CM at indicated concentrations for 24 h. The luciferase activity was then measured 24 h later with normalization to the activity of the β-galactosidase. Values are averages of three determinations with the standard deviations indicated by error bars. **P<0.01 compared to the control cells without niclosamide treatment.CC BY · PubMed Central
Figure 2 Effects of niclosamide on Wnt/β-catenin signaling in cancer cells. (A) Prostate cancer PC-3 and breast cancer MDA-MB-231 cells in 6-well plates were treated with niclosamide at the indicated concentrations for 24 h. The levels of cytosolic free β-catenin were then examined by GST-E-cadherin binding assay. (B) Prostate cancer PC-3 and breast cancer MDA-MB-231 cells in 24-well plates were transiently transfected with the TOPFlash luciferase construct and β-galactosidase-expressing vector in each well. After 24 h incubation, cells were treated with 2.4 µM niclosamide. The luciferase activity was then measured 24 h later with normalization to the activity of the β-galactosidase. Values are the average of triple determinations with the s.d. indicated by error bars. **P<0.01 compared to the control cells.
Figure 2 Effects of niclosamide on Wnt/β-catenin signaling in cancer cells. (A) Prostate cancer PC-3 and breast cancer MDA-MB-231 cells in 6-well plates were treated with niclosamide at the indicated concentrations for 24 h. The levels of cytosolic free β-catenin were then examined by GST-E-cadherin binding assay. (B) Prostate cancer PC-3 and breast cancer MDA-MB-231 cells in 24-well plates were transiently transfected with the TOPFlash luciferase construct and β-galactosidase-expressing vector in each well. After 24 h incubation, cells were treated with 2.4 µM niclosamide. The luciferase activity was then measured 24 h later with normalization to the activity of the β-galactosidase. Values are the average of triple determinations with the s.d. indicated by error bars. **P<0.01 compared to the control cells.CC BY · PubMed Central
Figure 3 Effects of niclosamide on expression of LRP6, phosph-LRP6 (p-LRP6), Dvl2, Axin2 and Cyclin D1 in cancer cells. (A) DU145 and PC3 prostate cancer cells and MDA-MB-231 and T-47D breast cancer cells in 6-well plates were treated with niclosamide at indicated concentrations for 24 h. The total cellular levels of LRP6, p-LRP6, Dvl2, Axin2 and Cyclin D1were then examined. All the samples were also probed with anti-actin antibody to verify equal loading. (B) PC-3 and DU145 prostate cancer cells and MDA-MB-231 and T-47D breast cancer cells in 6-well plates were treated with niclosamide at indicated concentrations for 24 h. The cytosolic levels of Dvl2 were then examined. All the samples were also probed with anti-actin antibody to verify equal loading.
Figure 3 Effects of niclosamide on expression of LRP6, phosph-LRP6 (p-LRP6), Dvl2, Axin2 and Cyclin D1 in cancer cells. (A) DU145 and PC3 prostate cancer cells and MDA-MB-231 and T-47D breast cancer cells in 6-well plates were treated with niclosamide at indicated concentrations for 24 h. The total cellular levels of LRP6, p-LRP6, Dvl2, Axin2 and Cyclin D1were then examined. All the samples were also probed with anti-actin antibody to verify equal loading. (B) PC-3 and DU145 prostate cancer cells and MDA-MB-231 and T-47D breast cancer cells in 6-well plates were treated with niclosamide at indicated concentrations for 24 h. The cytosolic levels of Dvl2 were then examined. All the samples were also probed with anti-actin antibody to verify equal loading.CC BY · PubMed Central
Figure 4 Effects of niclosamide on LRP6 expression and degradation. (A) PC-3 cells were incubated with 10 µg/ml of cycloheximide in the presence of Niclosamide (1.2 µM) or vehicle for 0, 3, 6, 10 or 24 h. Cells were then harvested, and the level of endogenous LRP6 was examined by Western blotting. Samples were also probed with anti-actin antibody to verify equal loading. The pixels for each band were measured, normalized and plotted. Data are mean values of three independent experiments with the SD values indicated by error bars. *P<0.05, **P<0.01 versus corresponding control value. (B) PC-3 and MDA-MB-231 cells in 6-well plates were treated with niclosamide for 24 h. The cells were then harvested and LRP6 mRNA levels were determined by real-time RT-PCR and normalized to the message levels of GAPDH mRNA. All the values are the average of triple determinations with the s.d. indicated by error bars.
Figure 4 Effects of niclosamide on LRP6 expression and degradation. (A) PC-3 cells were incubated with 10 µg/ml of cycloheximide in the presence of Niclosamide (1.2 µM) or vehicle for 0, 3, 6, 10 or 24 h. Cells were then harvested, and the level of endogenous LRP6 was examined by Western blotting. Samples were also probed with anti-actin antibody to verify equal loading. The pixels for each band were measured, normalized and plotted. Data are mean values of three independent experiments with the SD values indicated by error bars. *P<0.05, **P<0.01 versus corresponding control value. (B) PC-3 and MDA-MB-231 cells in 6-well plates were treated with niclosamide for 24 h. The cells were then harvested and LRP6 mRNA levels were determined by real-time RT-PCR and normalized to the message levels of GAPDH mRNA. All the values are the average of triple determinations with the s.d. indicated by error bars.CC BY · PubMed Central
Figure 5 Effects of niclosamide on cancer cell apoptosis and proliferation. (A) Cancer cells were treated with niclosamide at the indicated concentrations for 24 h. Floating and attached cells were combined for apoptosis detection by the Cell Death ELISA kit from Roche Diagnostics Corporation for histone-associated DNA fragments as described in Materials and Methods. (B) Cancer cells in 96-well plates were treated with niclosamide for 72 h. Cell viability was measured by the Cell Titer Glo Assay system. All the values are the average of triplicate determinations with the s.d. indicated by error bars. **P<0.01 versus corresponding control value.
Figure 5 Effects of niclosamide on cancer cell apoptosis and proliferation. (A) Cancer cells were treated with niclosamide at the indicated concentrations for 24 h. Floating and attached cells were combined for apoptosis detection by the Cell Death ELISA kit from Roche Diagnostics Corporation for histone-associated DNA fragments as described in Materials and Methods. (B) Cancer cells in 96-well plates were treated with niclosamide for 72 h. Cell viability was measured by the Cell Titer Glo Assay system. All the values are the average of triplicate determinations with the s.d. indicated by error bars. **P<0.01 versus corresponding control value.CC BY · PubMed Central

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Metadata

Authors
Lu, W., Lin, C., Roberts, M. J., Waud, W. R., Piazza, G. A., & Li, Y.
Affiliations
  • Southern Research Institute (US)
  • University of South Alabama (US)
Corresponding author
Li, Yonghe
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2011-12-16
Volume
6
Issue
12
Pages
e29290
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0029290
PMC
PMC3241710
OpenAlex
W2007145040
Cited by
230
References
50
Citations by year
2026: 14 · 2025: 13 · 2024: 16 · 2023: 10 · 2022: 12 · 2021: 18 · 2020: 22 · 2019: 19 · 2018: 17 · 2017: 20 · 2016: 21 · 2015: 21 · 2014: 14 · 2013: 8 · 2012: 5
FWCI
3.51
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • University of Rochester
  • National Institutes of Health (RO1CA124531)
  • National Cancer Institute (R01CA124531)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Wnt/β-catenin signaling in development and cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Hedgehog Signaling Pathway Studies
  • Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
Keywords
niclosamide, LRP6, Wnt/β-catenin signaling, Wnt co-receptor, protein degradation, prostate cancer, breast cancer, apoptosis, anticancer drug repurposing, antihelminthic drugs, chemoprevention
MeSH terms
Dishevelled Proteins, Breast Neoplasms, Drug Screening Assays, Antitumor, Female, Humans, Male, Neoplasm Proteins, Neoplasms, Niclosamide, Phosphoproteins, Phosphorylation, Prostatic Neoplasms, Apoptosis, Cell Line, Tumor, Adaptor Proteins, Signal Transducing, Cell Proliferation, Wnt Proteins, beta Catenin, HEK293 Cells, Proteolysis, Wnt Signaling Pathway, Low Density Lipoprotein Receptor-Related Protein-6, Wnt3A Protein

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