title: Glucosamine inhibits epidermal growth factor-induced proliferation and cell-cycle progression in retinal pigment epithelial cells.
abstract: OBJECTIVE
  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.
authors.affiliation: Graduate Institute of Medical Science, National Defense Medical Center, Taipei, Taiwan, Republic of China.
authors.forename: Chang-Min
authors.initials: CM
authors.lastname: Liang
authors.forename: Ming-Cheng
authors.initials: MC
authors.lastname: Tai
authors.forename: Yun-Hsiang
authors.initials: YH
authors.lastname: Chang
authors.forename: Yi-Hao
authors.initials: YH
authors.lastname: Chen
authors.forename: Ching-Long
authors.initials: CL
authors.lastname: Chen
authors.forename: Ming-Wei
authors.initials: MW
authors.lastname: Chien
authors.forename: Jiann-Torng
authors.initials: JT
authors.lastname: Chen
mesh_terms: D002453:Cell Cycle / Q000187:drug effects*; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D000091342:Endoplasmic Reticulum Chaperone BiP; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005944:Glucosamine / Q000494:pharmacology*; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology*; D050796:STAT3 Transcription Factor / Q000378:metabolism; D013312:Stress, Physiological / Q000187:drug effects; D015533:Transcriptional Activation / Q000187:drug effects; D051176:beta Catenin / Q000378:metabolism
chemical_list: D016213:Cyclins; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D051176:beta Catenin; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D005944:Glucosamine