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Inhibition of the Sodium/Potassium ATPase Impairs N-Glycan Expression and Function

¹ Ontario Cancer Institute, Princess Margaret Hospital, ² Department of Medical Biophysics, University of Toronto, ³ Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, and ⁴ The Hospital for Sick Children, Toronto, Ontario, Canada; and ⁵ University of New Hampshire, Durham, New Hampshire

Requests for reprints: Aaron D. Schimmer, 610 University Avenue, Toronto, ON, Canada M5G 2M9. Phone: 416-946-2838; Fax: 416-946-6546; E-mail: aaron.schimmer{at}utoronto.ca.

Key Words: cell migration and invasion • chemical biology • N-glycosylation

Aberrant N-linked glycans promote the malignant potential of cells by enhancing the epithelial-to-mesenchymal transition and the invasive phenotype. To identify small molecule inhibitors of N-glycan biosynthesis, we developed a chemical screen based on the ability of the tetravalent plant lectin L-phytohemagglutinin (L-PHA) to bind and crosslink surface glycoproteins with β1,6GlcNAc-branched complex type N-glycans and thereby induce agglutination and cell death. In this screen, Jurkat cells were treated with a library of off-patent chemicals (n = 1,280) to identify molecules that blocked L-PHA–induced death. The most potent hit from this screen was the cardiac glycoside (CG) dihydroouabain. In secondary assays, a panel of CGs was tested for their effects on L-PHA–induced agglutination and cell death. All of the CGs tested inhibited L-PHA–induced death in Jurkat cells, and the most potent CG tested was digoxin with an EC₅₀ of 60 ± 20 nmol/L. Digoxin also increased the fraction of some concanavalin A–binding N-glycans. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, digoxin specifically increased GlcNAc₁Man₃GlcNAc₂Fuc₁ and GlcNAc₂Man₃GlcNAc₂Fuc₁ oligosaccharides demonstrating an impairment of the N-glycan pathway. Consistent with this effect on the N-glycan pathway, digoxin inhibited N-glycosylation–mediated processes of tumor cell migration and invasion. Furthermore, digoxin prevented distant tumor formation in two mouse models of metastatic prostate cancer. Thus, taken together, our high throughput screen identified CGs as modifiers of the N-glycan pathway. These molecules can be used as tools to better understand the role of N-glycans in normal and malignant cells. Moreover, these results may partly explain the anticancer effect of CGs in cardiovascular patients. [Cancer Res 2008;68(16):6688–96]

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				C. D. Simpson, I. A. Mawji, K. Anyiwe, M. A. Williams, X. Wang, A. L. Venugopal, M. Gronda, R. Hurren, S. Cheng, S. Serra, et al. Inhibition of the Sodium Potassium Adenosine Triphosphatase Pump Sensitizes Cancer Cells to Anoikis and Prevents Distant Tumor Formation Cancer Res., April 1, 2009; 69(7): 2739 - 2747. [Abstract] [Full Text] [PDF]

				Correction: Cardiac Glycosides Impair the N-Glycosylation Pathway Cancer Res., September 15, 2008; 68(18): 7692 - 7692. [Full Text] [PDF]