Abstract
Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.
Highlights
From the ‡Department of Chemistry and Biomolecular Sciences, Macquarie University, NSW 2109, Australia; §Children’s Cancer Institute Australia, Lowy Cancer Research Centre, UNSW, Randwick, NSW 2031, Australia; ¶Australian Centre for Nanomedicine, UNSW, Sydney, 2052, Australia; ʈGraduate School of Advanced Sciences of Matter, Hiroshima University, Hiroshima 739-8530, Japan
We have previously described the selection and characterization of T-cell acute lyphoblastic leukemia cells selected for resistance to an epothilone B analog, Z12, 13-desoxyepothilone B1 [5, 6]
Glycans were released from cell membrane glycoproteins by PNGase F and were analyzed by LC-ESI MS and MS/MS as
Summary
Well as decreased membrane localization, thereby sensitizing MCF7-ADR cells to docetaxel [8]. These two reports focused on the relative alteration of glycan structures using lectin staining or gel migration changes on SDS-PAGE, but did not identify the actual glycan structures associated with these drug resistance mechanisms. All Nglycans have a common core structure, Man␣1– 6(Man␣1– 3)Man1– 4GlcNAc1– 4GlcNAc1-, and are classified into three types: [1] high-mannose type, in which only Man residues are attached to both of the Man␣1– 6 and Man␣1–3 arms of the core structure; [2] hybrid type, in which Man residues are attached to only the Man␣1– 6 arm, an “antennae” structure produced through the action of N-acetylglucosaminyltransferases being attached to the Man␣1–3 arm; and [3] complex type, in which the “antennae” structure is attached to both the Man␣1– 6 and Man␣1–3 arms. Alterations to the glycans attached to cell membrane proteins may contribute to the phenotype associated with dEpoB resistance
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