Amprenavir Mitigates Pepsin-Induced Transcriptomic Changes in Normal and Precancerous Esophageal Cells

Background: Gastroesophageal reflux disease (GERD) is a chronic condition linked to inflammatory and cancerous alterations in esophageal epithelium. Despite the widespread use of acid suppressing proton pump inhibitors (PPIs), the incidence of esophageal adenocarcinoma (EAC) keeps increasing, underscoring the possible importance of nonacidic reflux components like pepsin in cancer development. Pepsin, a therapeutic target for GERD, has been associated with epithelial damage and metaplasia. Methods: The goal of this study was to investigate the effect of pepsin, at a transcriptional level, and to assess the potential therapeutic value of amprenavir, as a pepsin inhibitor, in vitro. Human esophageal cell lines, immortalized from normal (Het-1A) and Barrett’s esophagus (BAR-T), were exposed to pH 7.0 buffer containing pepsin and/or amprenavir for 1 hour (n=3). RNA-seq was conducted to detect differentially expressed genes (DEGs) with FDR ≤ 0.05 and |log2 fold change| ≥ 0.375, and Ingenuity Pathway Analysis (IPA) was performed to identify the top significantly biological pathways. Results: Pepsin exposure caused notable transcriptomic alterations in both Het-1A and BAR-T cells, specifically impacting mitochondrial function, oxidative phosphorylation, and pathways associated with epithelial integrity, cell signaling, and inflammatory responses. Amprenavir alleviated several of these effects, indicating a protective function in epithelial impairment resulting from peptic injury. Of interest, Het-1A and BAR-T cells demonstrated different reactions to pepsin treatment; BAR-T cells revealed more pronounced changes in pathways associated with wound healing, chronic epithelial formation, and repair, whereas Het-1A cells showed more substantial alterations in systemic damage and mitochondrial pathways. Conclusion: The results of this study underscore the detrimental impact of pepsin on esophageal epithelial cells despite PPI therapy and highlight amprenavir as a promising therapeutic option to mitigate pepsin-induced epithelial injury. Further research to validate these in vitro findings is warranted to assess the efficacy of amprenavir for GERD-related damage and carcinogenesis. RNA-seq was performed on two cell lines, Het-1A and BAR-T, under three conditions: "Control," "Pepsin," and "Pepsin+APR," with three replicates per condition, resulting in a total of 18 samples.