Inhibition of the sodium/potassium ATPase impairs N-glycan expression and function.

Beheshti Zavareh R, Lau KS, Hurren R, Datti A, Ashline DJ, Gronda M, Cheung P, Simpson CD, Liu W, Wasylishen AR, Boutros PC, Shi H, Vengopal A, Jurisica I, Penn LZ, Reinhold VN, Ezzat S, Wrana J, Rose DR, Schachter H, Dennis JW, Schimmer AD
Cancer Res. 2008 68 (16): 6688-97

PMID: 18701493 · DOI:10.1158/0008-5472.CAN-07-6833

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 beta1,6GlcNAc-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 EC(50) 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 GlcNAc(1)Man(3)GlcNAc(2)Fuc(1) and GlcNAc(2)Man(3)GlcNAc(2)Fuc(1) 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.

MeSH Terms (22)

Animals Cell Movement Cell Survival Combinatorial Chemistry Techniques Digitalis Glycosides Enzyme Inhibitors Glycopeptides Glycosylation Humans Male Mice Mice, SCID Neoplasms Phytohemagglutinins Polysaccharides Reverse Transcriptase Polymerase Chain Reaction RNA, Small Interfering Sodium-Potassium-Exchanging ATPase Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Transfection Tumor Cells, Cultured Wound Healing

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