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

Abstract

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 B1,6GlcNAc-branched complex type N-glycans and thereby induce agglutination and cell death. In this screen, Jurkat cells were treated with a library of offpatent 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-PHAinduced 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 F 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. [Cancer Res 2008;68(16):6688-97]

The ubiquitin-activating enzyme E1 as a therapeutic target for the treatment of leukemia and multiple myeloma

“…12,17 The cDNAs, encoding GRP78, HSP70, and 18S rRNA, were amplified with the following primer pairs: GRP78, 5Ј-TAG CGT ATG GTG CTG CTG TC-3Ј (forward) and 5Ј-TTT GTC AGG GGT CTT TCA CC-3Ј (reverse); HSP70, 5Ј-CAA GAT CAC CAT CAC CAA CG-3Ј (forward) and 5Ј-GCT CAA ACT CGT CCT TCT CG-3Ј (reverse); 18S rRNA, 5Ј-AGG AAT TGA CGG AAG GGC AC-3Ј (forward) and 5Ј-GGA CAT CTAAGG GCA TCA CA-3Ј (reverse). Relative mRNA expression was determined with the ⌬⌬ CT normalization as previously described.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

“…Significant differences between profiles obtained by MALDI from patients and controls but patient sample too small to claim biomarkers (Kirmiz, et al, 2007b) Colon carcinoma HT29 cells Changes in activity of GlcNAc transferases III and IV but not V Hepatocellular carcinoma Serum "Enzymatic", TOF/TOF (DHB), N-glycans (per-Me) Development of peak selection algorithm to differentiate hepatocellular carcinoma and chronic liver disease (Varghese, et al, 2008) (Zavareh, et al, 2008) Lung cancer Human serum PNGase F, QIT-TOF (THAP), Nglycans Method development and identification of differences in core fucosylation of complex N-glycans in lung cancer Metastatic melanoma α 3 β 1 and α v β 3 integrin PNGase F (in gel), TOF (DHB), N-glycans From WM9 and WM293 cell lines. Heavily sialylated and fucosylated glycans plus Gal-GlcNAc extensions (Kremser, et al, 2008) Ovarian cancer Serum glycoproteins PNGase F (in-gel), TOF (DHB), ESI HPLC (2-AB)…”

Inhibition of the Sodium Potassium Adenosine Triphosphatase Pump Sensitizes Cancer Cells to Anoikis and Prevents Distant Tumor Formation

“…Male severe combined immunodeficient mice (SCID) were obtained from and housed at the Ontario Cancer Institute animal colony, kept in laminar-flow cage racks under standardized environmental conditions with access to food and water ad libitum. Distant tumor formation was assessed using our previously described model (10)(11)(12). A single common threshold was applied to identify and measure fluorescence in each organ.…”