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©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.
γ-aminobutyric acid B2 receptor: A potential therapeutic target for cholangiocarcinoma in patients with diabetes mellitus
Charupong Saengboonmee, Supannika Sorin, Sakkarn Sangkhamanon, Surang Chomphoo, Somsiri Indramanee, Wunchana Seubwai, Kanyarat Thithuan, Ching-Feng Chiu, Seiji Okada, Marie-Claude Gingras, Sopit Wongkham
Charupong Saengboonmee, Supannika Sorin, Somsiri Indramanee, Kanyarat Thithuan, Sopit Wongkham, Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
Charupong Saengboonmee, Supannika Sorin, Sakkarn Sangkhamanon, Somsiri Indramanee, Wunchana Seubwai, Sopit Wongkham, Center for Translational Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
Charupong Saengboonmee, Supannika Sorin, Sakkarn Sangkhamanon, Somsiri Indramanee, Wunchana Seubwai, Sopit Wongkham, Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen 40002, Thailand
Sakkarn Sangkhamanon, Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
Surang Chomphoo, Department of Anatomy, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
Wunchana Seubwai, Department of Forensic Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
Ching-Feng Chiu, Graduate Institute of Metabolism and Obesity Sciences, Taipei Medical University, Taipei 11031, Taiwan
Seiji Okada, Division of Hematopoiesis, Joint Research Center for Human Retrovirus Infection, Kumamoto University, Kumamoto 860-0811, Japan
Marie-Claude Gingras, Human Genome Sequencing Center, Department of Human and Molecular Genetics, Baylor College of Medicine, Houston, TX 77030, United States
Marie-Claude Gingras, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX 77030, United States
Author contributions: Saengboonmee C, Seubwai W, Gingras MC, and Wongkham S conceptualized the research; Saengboonmee C, Sorin S, Sangkhamanon S, Indramanee S, Chomphoo S, and Thithuan K designed and performed the experiments; Saengboonmee C, Sorin S, Sangkhamanon S, Chomphoo S, Seubwai W, Chiu CF, Okada S, Gingras MC, and Wongkham S analyzed the data; Okada S and Gingras MC contributed research resources and analysis tools; Saengbonmee C and Sorin S wrote the manuscript draft; Saengboonmee C, Chiu CF, Okada S, Gingras MC, and Wongkham S reviewed and edited the manuscript; and all authors read and approved the final version of the manuscript.
Supported by the Research Grant for Young Talented Scholars, National Research Council of Thailand, No. N41A640108.
Institutional review board statement: The protocol of this study has been reviewed and approved by the Khon Kaen University Ethics Committee for Human Research (approval No. HE641441), based on the Declaration of Helsinki and the ICH-Good Clinical Practice Guidelines.
Informed consent statement: Written informed consent was obtained from all participants.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
https://creativecommons.org/Licenses/by-nc/4.0/ Corresponding author: Charupong Saengboonmee, MD, PhD, Doctor, Lecturer, Department of Biochemistry, Faculty of Medicine, Khon Kaen University, 123 Mitraphap Highway, Khon Kaen 40002, Thailand. charusa@kku.ac.th
Received: February 23, 2023
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
BACKGROUND
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.
AIM
To investigate the effects of hyperglycemia on GABBR2 expression and the potential use of GABBR2 as a CCA therapeutic target.
METHODS
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.
RESULTS
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.
CONCLUSION
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.
