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©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.
Expression of the methylcytosine dioxygenase ten-eleven translocation-2 and connexin 43 in inflammatory bowel disease and colorectal cancer
Mohammad El-Harakeh, Jessica Saliba, Kawthar Sharaf Aldeen, May Haidar, Layal El Hajjar, Mireille Kallassy Awad, Jana G Hashash, Margret Shirinian, Marwan El-Sabban
Mohammad El-Harakeh, Kawthar Sharaf Aldeen, May Haidar, Layal El Hajjar, Marwan El-Sabban, Department of Anatomy, Cell Biology and Physiological Sciences, Faculty of Medicine, American University of Beirut, Beirut 1107, Lebanon
Mohammad El-Harakeh, Mireille Kallassy Awad, UR GPF Laboratory of Biodiversity and Functional Genomics, Faculty of Science, Université Saint-Joseph de Beyrouth, Beirut 1107, Lebanon
Jessica Saliba, Department of Biology, Faculty of Sciences, Lebanese University, Beirut 1533, Lebanon
Jessica Saliba, Department of Public Health, Faculty of Health Sciences, University of Balamand, Dekwaneh, Sin el Fil 1552, Lebanon
Jana G Hashash, Department of Internal Medicine, Division of Gastroenterology and Hepatology, Faculty of Medicine, American University of Beirut, Beirut 1107, Lebanon
Margret Shirinian, Department of Experimental Pathology, Immunology and Microbiology, Faculty of Medicine, American University of Beirut, Beirut 1107, Lebanon
Author contributions: El-Harakeh M, Saliba J, Haidar M, and Sharaf Aldeen K equally contributed to the study; El-Sabban M conceived and designed the study; Hashash JG, Awad MK, Shirinian M, and El-Sabban M provided the material and resources for the experiments; El-Harakeh M, Haidar M, Sharaf Aldeen K, and El Hajjar L performed the experiments; Saliba J and El-Harakeh M analyzed the data and wrote the manuscript; Shirinian M and Hashash JG critically reviewed the manuscript; Saliba J and El-Sabban M revised the manuscript; All authors read and approved the final version of the manuscript.
Institutional review board statement: Archived formaldehyde-fixed paraffin-embedded (FFPE) colon blocks (ulcerative colitis, sporadic colon adenocarcinoma and normal) were obtained from the American University of Beirut Medical Center (AUBMC). All patients’ identifiers were kept confidential and all samples were anonymous, hence exempted from ethics committee approval.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the American University of Beirut, No. 18-03-476.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: Raw data are available upon reasonable request.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
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: Marwan El-Sabban, PhD, Professor, Department of Anatomy, Cell Biology and Physiological Sciences, Faculty of Medicine, American University of Beirut, Bliss Street, Beirut 1107, Lebanon. me00@aub.edu.lb
Received: February 6, 2022
Peer-review started: February 6, 2022
First decision: April 10, 2022
Revised: May 6, 2022
Accepted: June 13, 2022
Article in press: June 13, 2022
Published online: October 28, 2022
BACKGROUND
Inflammatory bowel disease (IBD) constitutes a substantial risk factor for colorectal cancer. Connexin 43 (Cx43) is a protein that forms gap junction (GJ) complexes involved in intercellular communication, and its expression is altered under pathological conditions, such as IBD and cancer. Recent studies have implicated epigenetic processes modulating DNA methylation in the pathogenesis of diverse inflammatory and malignant diseases. The ten-eleven translocation-2 (TET-2) enzyme catalyzes the demethylation, hence, regulating the activity of various cancer-promoting and tumor-suppressor genes.
AIM
To investigate Cx43 and TET-2 expression levels and presence of 5-hydroxymethylcytosine (5-hmC) marks under inflammatory conditions both in vitro and in vivo.
METHODS
TET-2 expression was evaluated in parental HT-29 cells and in HT-29 cells expressing low or high levels of Cx43, a putative tumor-suppressor gene whose expression varies in IBD and colorectal cancer, and which has been implicated in the inflammatory process and in tumor onset. The dextran sulfate sodium-induced colitis model was reproduced in BALB/c mice to evaluate the expression of TET-2 and Cx43 under inflammatory conditions in vivo. In addition, archived colon tissue sections from normal, IBD (ulcerative colitis), and sporadic colon adenocarcinoma patients were obtained and evaluated for the expression of TET-2 and Cx43. Expression levels were reported at the transcriptional level by quantitative real-time polymerase chain reaction, and at the translational level by Western blotting and immunofluorescence.
RESULTS
Under inflammatory conditions, Cx43 and TET-2 expression levels increased compared to non-inflammatory conditions. TET-2 upregulation was more pronounced in Cx43-deficient cells. Moreover, colon tissue sections from normal, ulcerative colitis, and sporadic colon adenocarcinoma patients corroborated that Cx43 expression increased in IBD and decreased in adenocarcinoma, compared to tissues from non-IBD subjects. However, TET-2 expression and 5-hmC mark levels decreased in samples from patients with ulcerative colitis or cancer. Cx43 and TET-2 expression levels were also investigated in an experimental colitis mouse model. Interestingly, mice exposed to carbenoxolone (CBX), a GJ inhibitor, had upregulated TET-2 levels. Collectively, these results show that TET-2 levels and activity increased under inflammatory conditions, in cells downregulating gap junctional protein Cx43, and in colon tissues from mice exposed to CBX.
CONCLUSION
These results suggest that TET-2 expression levels, as well as Cx43 expression levels, are modulated in models of intestinal inflammation. We hypothesize that TET-2 may demethylate genes involved in inflammation and tumorigenesis, such as Cx43, potentially contributing to intestinal inflammation and associated carcinogenesis.
Core Tip: Chronic inflammation of the colon is a risk factor for colorectal cancer. Intestinal inflammation has been associated with reduced gap junction-mediated intercellular communication and downregulation connexins [mainly connexin 43 (Cx43)]. The involvement of the demethylating enzyme ten-eleven translocation-2 (TET-2) (methylcytosine dioxygenase) in the inflammatory process has motivated the investigation of Cx43 and TET-2 expression in colitis and colorectal carcinoma. In vitro and in vivo data report on the upregulation of Cx43 in inflammatory states and a downregulation of TET-2. In human samples of colon adenocarcinoma, both TET-2 and Cx43 were downregulated, potentially implicating them in the malignant transformation of inflamed intestinal tissues.
