Glucosamine inhibits epidermal growth factor-induced proliferation and cell-cycle progression in retinal pigment epithelial cells
Glucosamine inhibits epidermal growth factor-induced proliferation and cell-cycle progression in retinal pigment epithelial cells
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发表时间：
2010-12
期刊：
影响因子：
2.2
通讯作者：
C. Liang;Ming‐Cheng Tai;Yun-Hsiang Chang;Yi-Hao Chen;Ching-Long Chen;Ming-Wei Chien;Jiann-Torng Chen
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作者：
C. Liang;Ming‐Cheng Tai;Yun-Hsiang Chang;Yi-Hao Chen;Ching-Long Chen;Ming-Wei Chien;Jiann-Torng Chen
目的探讨氨基葡萄糖（GlcN）对表皮生长因子（EGF）诱导的视网膜色素上皮细胞增殖的影响及其机制。方法采用4-[3-（4-碘苯基）-2-（4-硝基苯基）-2H-5-四唑基]-1，3-苯二磺酸盐（WST-1）比色法和细胞计数法，检测人视网膜色素上皮细胞株（ARPE-19）的增殖情况。结果在人供体细胞中用羧基荧光素二乙酸琥珀酰亚胺酯细胞增殖试验（CFSE）细胞增殖试验证实。在ARPE-19细胞中，通过流式细胞术测定细胞周期进程;通过蛋白质印迹法测定细胞周期调节剂和热休克蛋白90（Hsp 90）的蛋白质水平;使用L-菜豆凝集素凝集素结合试验评估N-聚糖的水平和分支;通过蛋白质印迹法检查N-聚糖对EGF受体（EGFR）的调节。结果GlcN呈剂量依赖性抑制视网膜色素上皮（RPE）增殖。在EGF诱导的细胞周期进程中，GlcN引起G1-S和G2-M转换的延迟，而不影响细胞活力。GlcN调节EGFR上N-聚糖的水平和分支，抑制EGFR的磷酸化，并减少细胞外信号调节激酶、苏氨酸/苏氨酸蛋白激酶和信号转导和转录激活因子3（STAT 3）的磷酸化。GlcN对内质网中非特异性应激的Hsp 90、Grp 78和转录因子CHOP/GADD 153标记物的表达仅有轻微影响。结论GlcN能有效抑制体外培养的RPE细胞增殖。这种作用似乎是通过修饰EGFR上的N-聚糖实现的。进一步研究GlcN作为预防和治疗RPE介导的眼部增殖性疾病（如增殖性玻璃体视网膜病变和其他EGF依赖性增殖性细胞生长疾病）的潜在药物的作用是必要的。
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 G1–S and G2–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.