Inhibition of the sodium/potassium ATPase impairs N-glycan expression and function.
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.
Beheshti Zavareh
Reza
R
Ontario Cancer Institute, Princess Margaret Hospital, Canada.
Lau
Ken S
KS
Hurren
Rose
R
Datti
Alessandro
A
Ashline
David J
DJ
Gronda
Marcela
M
Cheung
Pam
P
Simpson
Craig D
CD
Liu
Wei
W
Wasylishen
Amanda R
AR
Boutros
Paul C
PC
Shi
Hui
H
Vengopal
Amudha
A
Jurisica
Igor
I
Penn
Linda Z
LZ
Reinhold
Vern N
VN
Ezzat
Shereen
S
Wrana
Jeff
J
Rose
David R
DR
Schachter
Harry
H
Dennis
James W
JW
Schimmer
Aaron D
AD
D000818:Animals; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D020650:Combinatorial Chemistry Techniques; D004071:Digitalis Glycosides / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006020:Glycopeptides / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008297:Male; D051379:Mice; D016513:Mice, SCID; D009369:Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D010835:Phytohemagglutinins / Q000494:pharmacology; D011134:Polysaccharides / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection; D014407:Tumor Cells, Cultured; D014945:Wound Healing
D004071:Digitalis Glycosides; D004791:Enzyme Inhibitors; D006020:Glycopeptides; D010835:Phytohemagglutinins; D011134:Polysaccharides; D034741:RNA, Small Interfering; D000254:Sodium-Potassium-Exchanging ATPase