Quantitative Proteomics Analysis Reveals Possible Anticancer Mechanisms of 5’-deoxy-5’-methylthioadenosine in Cholangiocarcinoma Cells
Kankamol Kerdkumthong, Sutthipong Nanarong, Sittiruk Roytrakul, Thanawat Pitakpornpreecha, Phonprapavee Tantimetta, Phanthipha Runsaeng, Sumalee Obchoei
Abstract
Cholangiocarcinoma (CCA) is an aggressive cancer originating from bile duct epithelium, particularly prevalent in Asian countries with liver fluke infections. Current chemotherapy for CCA often fails due to drug resistance, necessitating novel anticancer agents. This study investigates the potential of 5’-deoxy-5’-methylthioadenosine (MTA), a naturally occurring nucleoside, against CCA. While MTA has shown promise against various cancers, its effects on CCA remain unexplored. We evaluated MTA’s anticancer activity in CCA cell lines and drug-resistant sub-lines, assessing cell viability, migration, invasion, and apoptosis. The potential anticancer mechanisms of MTA were explored through proteomic analysis using LC-MS/MS and bioinformatic analysis. The results show a dose-dependent reduction in CCA cell viability, with enhanced effects on cancer cells compared to normal cells. Moreover, MTA inhibits growth, induces apoptosis, and suppresses cell migration and invasion.
Introduction
Cholangiocarcinoma (CCA) is an aggressive cancer that originates from the bile duct epithelium [1]. While it is considered rare globally, it is more prevalent in certain Asian countries, with the highest incidence in Thailand, where there is a high prevalence of the causative liver fluke infection [2]. Over the past few decades, both the incidence and mortality rates of CCA have significantly increased worldwide [3–5]. CCA tends to develop silently but exhibits a high level of invasiveness [6]. Consequently, it often remains asymptomatic in its early stages and is diagnosed at an advanced stage. Patients with metastatic CCA are poor candidates for surgery, and as a result, the common treatment for these patients is chemotherapy. Unfortunately, CCA shows a high resistance to chemotherapeutic drugs, leading to ineffective treatments and poor overall survival rates [7]. Even when a combination of chemotherapeutic drugs is used, the survival of patients with CCA remains poor. Therefore, there is an urgent to pursue new anticancer agents that are effective with minimal side effects.
Materials & methods
Chemicals and reagents
5´-deoxy-5´-methylthioadenosine (MTA), gemcitabine, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagents were purchased from Sigma (Sigma—Aldrich, St. Louis, MO, USA). Matrigel™ was purchased from BD Bioscience (BD Bioscience, Bedford, MA, USA).
Cell culture
Two human CCA cell lines, KKU-213A and KKU-213B, were established from tissues of Thai CCA patients [15]. An immortalized human cholangiocyte cell line (MMNK-1) was established as previously described [16]. These cell lines were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan). The gemcitabine-resistant cells, KKU-213A-GR and KKU-213B-GR, were established from the CCA parental cell lines, KKU-213A and KKU-213B, respectively, as previously described [17]. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, NY, USA) and 1% antibiotics/antimycotics (Capricorn Scientific), in a humidified atmosphere of 5% CO2 at 37°C.
Results
MTAP deficiency in CCA cell lines
The RT-qPCR analysis was conducted to examine the mRNA levels of MTAP in CCA cell lines and normal cell line. The results indicated a significant downregulation of MTAP expression in both CCA cell lines (KKU-213A and KKU-213B) when compared to the normal cell line (MMNK-1) (Fig 1B).
Discussion
In our current study, we assessed the effect of MTA on KKU-213A, KKU-213B, and their respective drug-resistant cell lines. Our data demonstrated that MTA treatment exerts a significant anticancer effect by inhibiting cell proliferation, migration, invasion, and inducing apoptosis in CCA cells. Notably, MTA treatment also augmented the anticancer efficacy of gemcitabine in drug-resistant cells. The results from proteomics analysis revealed that MTA effectively inhibited various proteins that have oncogenic properties, corroborating earlier findings.
Acknowledgments
The authors acknowledge the resources provided by the Central Equipment Center, Faculty of Science, Prince of Songkla University, specifically for the fluorescence microscope (cellSens Standard software, Olympus IX73 inverted microscope). The authors would also like to express gratitude to Miss Kawinnath Songsurin for her contribution to establishing the gemcitabine-resistant cell lines, KKU-213A-GR, and KKU-213B-GR.
Citation: Kerdkumthong K, Nanarong S, Roytrakul S, Pitakpornpreecha T, Tantimetta P, Runsaeng P, et al. (2024) Quantitative proteomics analysis reveals possible anticancer mechanisms of 5’-deoxy-5’-methylthioadenosine in cholangiocarcinoma cells. PLoS ONE 19(6): e0306060. https://doi.org/10.1371/journal.pone.0306060
Editor: Matias A. Avila, University of Navarra School of Medicine and Center for Applied Medical Research (CIMA), SPAIN
Received: February 20, 2024; Accepted: June 10, 2024; Published: June 26, 2024
Copyright: © 2024 Kerdkumthong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files. The MS proteomics data from this research have been deposited in the ProteomeXchange Consortium (http://www.proteomexchange.org/) through the jPOSTrepo partner (https://repository.jpostdb.org/entry/JPST002497), with dataset identifier PXD049119.
Funding: This research was supported by the National Science, Research and Innovation Fund (NSRF) and Prince of Songkla University (Ref. No. SCI6701024S) to Sumalee Obchoei. Kankamol Kerdkumthong was supported by a Prince of Songkla University-Ph.D. Scholarship (PSU_PHD2561-001). Phonprapavee Tantimetta was supported by a Prince of Songkla University-Ph.D. Scholarship (PSU_PHD2565-002). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0306060#sec001
