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Aberrant N-linked glycosylation promotes the malignant potential of cells leading to abnormal proliferation, adhesion and invasion. To identify small molecule N-glycosylation inhibitors, we developed a chemical screen based on the ability of the tetravalent plant lectin PHA (phytohemagglutinin) to bind and cross-link 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=2500) to identify molecules that blocked PHA-induced death as measured by MTS assay. The most potent hit from this screen was dihyroouabain.

Dihydrooubain belongs to the cardiac glycoside (CG) family of Na+/K+- ATPase inhibitors, so we tested a panel of CGs for their effects on PHA-induced agglutination and cell death. All of the CGs tested inhibited PHA-induced death in Jurkat cells and the most potent CG tested was digoxin with an EC50 of 60 ± 20 nM. As further evidence of the effects of digoxin on glycosylation, we tested whether digoxin inhibited the synthesis of new glycoproteins. Treatment with digoxin increased the fraction of high mannose N-glycans consistent with an effect of digoxin on blocking the N-glycosylation pathway. Analysis of the glycan profile of digoxin treated cells by MALD-TOF mass spectrometry revealed an increase in the abundance of GnM3Gn2F1 and Gn2M3Gn2F1 glycans that do not bind PHA. These data demonstrate that digoxin treatment leads to a block in the N-glycosylation pathway downstream of Golgi Manosidase II.

Increased expression of complex N-glycans affects the spreading and the migration of malignant cells. Therefore, we investigated the effect of digoxin treatment on these glycosylation-mediated processes. HeLa cells were treated with digoxin and cultured on fibronectin and collagen-coated wells to promote adhesion. At increasing times after culture, spreading was measured using automated fluorescent microscopy. Digoxin treated cells displayed increased spreading on fibronectin but not collagen coated surfaces. To assess the effect of digoxin on cell migration, HT1080 fibrosarcoma cells were treated with digoxin and buffer control. After treatment, cells were harvested and assessed for migratory ability in the scratch wound-healing experiment. Digoxin treatment decreased the migration of the HT1080 cells. Thus, treatment with digoxin converted malignant cells to a non-motile phenotype consistent with inhibition of complex N-glycan synthesis.

Taken together, a high throughput screen identified cardiac glycosides as inhibitors of the complex N-glycan synthesis. These molecules can be used as tools to better understand the role of glycosylation in normal and malignant cells. In addition, molecules based on digoxin may be leads for novel therapeutic agents for the treatment of malignancy.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA