Glucosamine inhibits epidermal growth factor-induced proliferation and cell-cycle progression in retinal pigment epithelial cells

PURPOSE: To investigate the effects and mechanisms of glucosamine (GlcN) on the proliferation of retinal pigment epithelial cells in response to epidermal growth factor (EGF). METHODS: Cell proliferation was measured in the human retinal pigment epithelial cell line (ARPE-19) cells with the 4-[3-(4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay and cell counting. The results were confirmed in human donor cells with the carboxyfluorescein diacetate succinimidyl ester cell proliferation assay (CFSE) cell proliferation assay. In ARPE-19 cells, cell-cycle progression was determined by flow cytometry; the protein levels of cell cycle regulators and heat shock protein 90 (Hsp90) were measured by western blotting; the levels and branching of N-glycans were assessed using the L-Phaseolus vulgaris agglutinin lectin-binding assay; and the modulation of N-glycans on EGF receptor (EGFR) was examined by western blotting. RESULTS: GlcN inhibited retinal pigment epithelium (RPE) proliferation in a dose-dependent manner. During cell-cycle progression induced by EGF, GlcN caused delays at the G(1)–S and G(2)–M transitions without affecting cell viability. GlcN modulated the level and branching of N-glycans on EGFR, suppressed phosphorylation of EGFR, and reduced phosphorylation of extracellular signal-regulated kinases, erine/threonine protein kinase, and the signal transducer and activator of transcription 3 (STAT3). GlcN had only minor effects on the expression of Hsp90, Grp78, and transcription factor CHOP/GADD 153 markers of nonspecific stress in the endoplasmic reticulum. CONCLUSIONS: GlcN effectively suppressed proliferation of RPE cells in vitro. This effect appeared to be achieved through modification of N-glycans on EGFR. Further research into the role of GlcN as a potential agent for the prevention and treatment of RPE-mediated ocular proliferative disorders, such as proliferative vitreoretinopathy, and other EGF-dependent proliferative cell-growth disorders, is warranted.