Carbon Monoxide as a Cancer-Selective Anti-Neoplastic Agent in Esophageal Epithelial Cells

Barrett's esophagus (BE) is a premalignant adaptation to chronic gastroesophageal reflux disease (GERD) and the principal precursor lesion of esophageal adenocarcinoma (EAC). Carbon monoxide (CO), an endogenous gaseous mediator generated by heme oxygenases, regulates cellular homeostasis, stress responses, and survival pathways; however, its role in BE progression and EAC remains poorly understood. We investigated the biological and transcriptional effects of increased CO bioavailability in cellular models representing distinct stages of disease progression. Non-transformed esophageal squamous epithelial cells (NES-G2T), BE cells (BAR-T), and EAC-derived OE19 and OE33 cells were exposed to the CO donor CORM-A1 or its CO-depleted structural control (iCORM-A1). Cellular metabolic activity was assessed by the MTT assay, apoptosis by Annexin V/PI flow cytometry, and transcriptomic alterations by RNA sequencing in BAR-T, OE19, and OE33 cells following CORM-A1 treatment. Transient CO exposure was well tolerated by NES-G2T cells, with no significant effects on metabolic activity or apoptosis. While BAR-T cells showed alterations in MTT-derived metabolic activity, they exhibited only limited apoptotic changes following CORM-A1 exposure, indicating that the reduced metabolic activity was not accompanied by significant apoptotic cell death. In contrast, CORM-A1 induced a reduction in metabolic activity and increased apoptotic cell death in EAC-derived OE19 and OE33 cells. At 500 μM, CORM-A1 reduced metabolic activity of both EAC cell lines below 75% of the untreated control and exerted a significantly greater reduction in metabolic activity than iCORM-A1, suggesting a contribution of CO release to the observed response. Flow cytometric analyses confirmed a noticeable apoptotic response to CORM-A1 in both EAC cell lines. RNA sequencing revealed marked, highly cell type-dependent transcriptional responses to CO exposure. Non-neoplastic BAR-T cells exhibited the most pronounced transcriptomic remodeling, predominantly affecting pathways related to epithelial differentiation, tissue homeostasis, and extracellular matrix organization. In contrast, OE33 and OE19 cells displayed more restricted transcriptional responses that preferentially involved growth factor signaling, cellular adaptation, inflammatory regulation, and tumor-microenvironment interactions. Collectively, these findings suggest that responsiveness to CO is shaped by the degree of neoplastic transformation, with BE mounting a broad adaptive transcriptional response, showing limited drug-induced apoptosis. However, malignant cells exhibit greater vulnerability to CORM-A1-induced programmed cell death despite undergoing more limited transcriptomic remodeling. These data identify CORM-A1-responsive pathways and CO signaling as potential therapeutic targets in EAC.