Published online Jul 28, 2023. doi: 10.3748/wjg.v29.i28.4416
Peer-review started: February 23, 2023
First decision: May 16, 2023
Revised: June 5, 2023
Accepted: July 5, 2023
Article in press: July 5, 2023
Published online: July 28, 2023
Processing time: 153 Days and 3.4 Hours
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 (P < 0.01). High GABBR2 expression was significantly associated with a poorer non-papillary histological subtype but with smaller sizes of CCA tumors (P < 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 (P < 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.
Core Tip: Diabetes mellitus is associated with an increased risk and progression of cholangiocarcinoma (CCA). The γ-aminobutyric acid (GABA) B2 receptor (GABBR2) was upregulated in CCA cells cultured in high glucose and in CCA tissues from patients with hyperglycemia. High GABBR2 expressions were significantly correlated with a non-papillary histotype and smaller sizes of CCA tumors. The treatment of baclofen, a GABA-B receptor agonist, significantly suppressed CCA cell proliferation and clonogenicity, suggesting that GABBR2 is a potential target for CCA treatment. Baclofen inhibited multiple kinases and signal transducers in CCA, resulting in downregulated downstream target proteins involving cell proliferation and suppression of CCA cell growth.