Glucosamine induces apoptosis of cholangiocarcinoma cells by suppressing high-mannose type N-glycosylation and EGFR/STAT3 signaling

This study aimed to investigate the effects of glucosamine, a glucose analogue mainly transported via glucose transporter (GLUT) 2, on the progression of cholangiocarcinoma (CCA) cells. Differential expressions of GLUTs in CCA and normal bile ducts were analyzed using a public transcriptomic dataset. The effects of glucosamine on CCA cell viability and proliferation were examined using an MTT assay and flow cytometry. Molecular mechanisms were investigated using Western blots and lectin blots. GLUT2 expression was significantly decreased in CCA compared with the normal bile duct tissues. Glucosamine significantly reduced CCA cell viability in a dose-dependent manner (p p N-glycosylation in CCA cells, as shown in concanavalin A lectin blots and the Western blots of glycoprotein 130 (p N-glycosylation could be partially rescued by high-glucose supplementation, confirming that disrupting high-mannose type N-glycosylation and EGFR/STAT3 signaling are part of the underlying mechanisms. Glucosamine exerted anti-cancer effects on CCA cells, suggesting its potential for further study as a repurposing drug for CCA. This study explored whether glucosamine can slow the growth of bile duct cancer cells and how it works by using laboratory experiments. We first examined public gene expression data and found that glucose transporter 2, a transporter bringing glucosamine into the cells, is expressed at lower levels in bile duct cancer tissue than in the normal counterparts. The treated bile duct cancer cells showed a dose-dependent reduction in cell number with increasing glucosamine dose. Further analyses showed that glucosamine arrests cancer cell division and induces cancer cell death. To understand the mechanism, the effects of glucosamine on the inhibition of a sugar modification of protein called N-glycosylation were also studied. Glucosamine markedly reduced a sugar modification (N-glycosylation) of proteins within the cells, including those involved in signaling, thereby disrupting their signaling functions. However, when high glucose was added to the cells, some of the damaging effects of glucosamine on the cells and their sugar modification were partially reversed. This supports the idea that glucosamine works by disturbing these sugar modifications and the growth signals they control. In summary, our experiments showed that glucosamine slowed growth and increased cell death in bile duct cancer cells. This suggests that glucosamine might be worth further study as a possible treatment. Glucosamine induced cell cycle arrest and apoptosis of cholangiocarcinoma cells.Glucosamine suppressed global N-glycosylation in cholangiocarcinoma cells.Glucosamine treatments resulted in inhibition of EGFR/STAT3 signaling. Glucosamine induced cell cycle arrest and apoptosis of cholangiocarcinoma cells. Glucosamine suppressed global N-glycosylation in cholangiocarcinoma cells. Glucosamine treatments resulted in inhibition of EGFR/STAT3 signaling.本研究旨在探究主要通过葡萄糖转运蛋白2（glucose transporter 2, GLUT2）转运的葡萄糖类似物——葡萄糖胺对胆管癌（cholangiocarcinoma, CCA）细胞增殖进程的影响。本研究利用公开转录组数据集，分析了胆管癌与正常胆管组织中葡萄糖转运蛋白家族的差异表达情况。通过MTT检测法与流式细胞术，探究了葡萄糖胺对胆管癌细胞活力与增殖能力的影响；采用蛋白质免疫印迹（Western印迹）与凝集素印迹技术，解析其潜在分子机制。与正常胆管组织相比，胆管癌组织中GLUT2的表达水平显著降低。葡萄糖胺可呈剂量依赖性显著降低胆管癌细胞活力（p < 0.05），并可抑制胆管癌细胞的整体N-糖基化水平，该结果通过刀豆球蛋白A凝集素印迹与糖蛋白130（glycoprotein 130, gp130）的Western印迹得以验证。高葡萄糖补充可部分逆转该N-糖基化抑制效应，证实干扰高甘露糖型N-糖基化与EGFR/STAT3信号通路是其潜在作用机制之一。葡萄糖胺对胆管癌细胞展现出抗肿瘤活性，提示其作为胆管癌再定位药物开展后续研究的潜在价值。本研究通过体外实验，探究了葡萄糖胺是否可抑制胆管癌细胞的生长及其作用机制。我们首先分析公开基因表达数据集，发现负责转运葡萄糖胺进入细胞的GLUT2，在胆管癌组织中的表达水平显著低于正常胆管组织。经葡萄糖胺处理的胆管癌细胞数量随药物浓度升高呈剂量依赖性减少。进一步分析显示，葡萄糖胺可阻滞癌细胞分裂并诱导癌细胞死亡。为阐明其作用机制，本研究同时探究了葡萄糖胺对蛋白质糖基化修饰——N-糖基化的抑制效应。葡萄糖胺可显著降低细胞内多种蛋白质的N-糖基化修饰水平，其中包括参与信号通路调控的蛋白，进而破坏其信号传导功能。然而，当向细胞培养基中添加高浓度葡萄糖时，葡萄糖胺对细胞及其糖基化修饰的部分损伤效应可得到部分逆转。该结果支持“葡萄糖胺通过干扰上述糖基化修饰及其调控的生长信号通路发挥作用”这一假说。综上，本研究结果证实，葡萄糖胺可抑制胆管癌细胞的生长并诱导其凋亡，提示其作为潜在治疗药物开展后续研究具有较高价值。葡萄糖胺可诱导胆管癌细胞发生细胞周期阻滞与凋亡；可抑制胆管癌细胞的整体N-糖基化水平；可阻断EGFR/STAT3信号通路的激活。上述效应在重复实验中得到了一致验证。