Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
Center for Translational Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.
World J Gastroenterol. 2023 Jul 28;29(28):4416-4432. doi: 10.3748/wjg.v29.i28.4416.
The association between diabetes mellitus (DM) and the increased risk and progression of cholangiocarcinoma (CCA) has been reported with unclear underlying mechanisms. Previous studies showed that γ-aminobutyric acid (GABA) B2 receptor (GABBR2) was upregulated in CCA cells cultured in high glucose (HG) conditions. Roles of GABA receptors in CCA progression have also been studied, but their association with DM and hyperglycemia in CCA remains unclarified.
To investigate the effects of hyperglycemia on GABBR2 expression and the potential use of GABBR2 as a CCA therapeutic target.
CCA cells, KKU-055 and KKU-213A, were cultured in Dulbecco Modified Eagle's Medium supplemented with 5.6 mmol/L (normal glucose, NG) or 25 mmol/L (HG) glucose and assigned as NG and HG cells, respectively. GABBR2 expression in NG and HG cells was investigated using real-time quantitative polymerase chain reaction and western blot. Expression and localization of GABBR2 in CCA cells were determined using immunocytofluorescence. GABBR2 expression in tumor tissues from CCA patients with and without DM was studied using immunohistochemistry, and the correlations of GABBR2 with the clinicopathological characteristics of patients were analyzed using univariate analysis. Effects of baclofen, a GABA-B receptor agonist, on CCA cell proliferation and clonogenicity were tested using the MTT and clonogenic assays. Phospho-kinases arrays were used to screen the affected signaling pathways after baclofen treatment, and the candidate signaling molecules were validated using the public transcriptomic data and western blot.
GABBR2 expression in CCA cells was induced by HG in a dose- and time-dependent manner. CCA tissues from patients with DM and hyperglycemia also showed a significantly higher GABBR2 expression compared with tumor tissues from those with euglycemia ( < 0.01). High GABBR2 expression was significantly associated with a poorer non-papillary histological subtype but with smaller sizes of CCA tumors ( < 0.05). HG cells of both tested CCA cell lines were more sensitive to baclofen treatment. Baclofen significantly suppressed the proliferation and clonogenicity of CCA cells in both NG and HG conditions ( < 0.05). Phospho-kinase arrays suggested glycogen synthase kinase 3 (GSK3), β-catenin, and the signal transducer and activator of transcription 3 (STAT3) as candidate signaling molecules under the regulation of GABBR2, which were verified in NG and HG cells of the individual CCA cell lines. Cyclin D1 and c-Myc, the common downstream targets of GSK3/β-catenin and STAT3 involving cell proliferation, were accordingly downregulated after baclofen treatment.
GABBR2 is upregulated by HG and holds a promising role as a therapeutic target for CCA regardless of the glucose condition.
糖尿病（DM）与胆管癌（CCA）风险增加和进展相关的关联已有报道，但潜在机制尚不清楚。先前的研究表明，在高葡萄糖（HG）条件下培养的 CCA 细胞中，γ-氨基丁酸（GABA）B2 受体（GABBR2）上调。GABA 受体在 CCA 进展中的作用也已被研究，但它们与 CCA 中的 DM 和高血糖的关联仍不清楚。
研究高血糖对 GABBR2 表达的影响，并探讨 GABBR2 作为 CCA 治疗靶点的潜力。
用含有 5.6mmol/L（正常葡萄糖，NG）或 25mmol/L（HG）葡萄糖的 Dulbecco 改良 Eagle 培养基培养 CCA 细胞 KKU-055 和 KKU-213A，并分别命名为 NG 和 HG 细胞。用实时定量聚合酶链反应和 Western blot 检测 NG 和 HG 细胞中 GABBR2 的表达。用免疫细胞荧光法检测 CCA 细胞中 GABBR2 的表达和定位。用免疫组织化学法检测有和无 DM 的 CCA 患者肿瘤组织中 GABBR2 的表达，并采用单因素分析分析 GABBR2 与患者临床病理特征的相关性。用 MTT 和集落形成实验检测 GABA-B 受体激动剂巴氯芬对 CCA 细胞增殖和集落形成的影响。用磷酸化激酶阵列筛选巴氯芬处理后的受影响信号通路，并通过公共转录组数据和 Western blot 验证候选信号分子。
HG 以剂量和时间依赖的方式诱导 CCA 细胞中 GABBR2 的表达。来自 DM 和高血糖患者的 CCA 组织与来自血糖正常患者的肿瘤组织相比，GABBR2 表达明显更高（<0.01）。高 GABBR2 表达与非乳头组织学亚型较差显著相关，但与 CCA 肿瘤的较小大小相关（<0.05）。两种测试的 CCA 细胞系的 HG 细胞对巴氯芬治疗更敏感。巴氯芬在 NG 和 HG 条件下均显著抑制 CCA 细胞的增殖和集落形成（<0.05）。磷酸化激酶阵列提示糖原合酶激酶 3（GSK3）、β-连环蛋白和信号转导和转录激活因子 3（STAT3）作为 GABBR2 调节下的候选信号分子，这在个别 CCA 细胞系的 NG 和 HG 细胞中得到了验证。涉及细胞增殖的 GSK3/β-连环蛋白和 STAT3 的共同下游靶标细胞周期蛋白 D1 和 c-Myc 也相应地下调。
HG 上调 GABBR2，无论葡萄糖条件如何，GABBR2 作为 CCA 的治疗靶点都具有很大的潜力。
