The Protease Inhibitor Amprenavir Protects against Pepsin-Induced Esophageal Epithelial Barrier Disruption and Cancer-Associated Changes

Abstract

Gastroesophageal reflux disease (GERD) significantly impacts patient quality of life and is a major risk factor for the development of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). Proton pump inhibitors (PPIs) are the standard-of-care for GERD and are among the most prescribed drugs in the world, but do not protect against nonacid components of reflux such as pepsin, or prevent reflux-associated carcinogenesis. We recently identified an HIV protease inhibitor amprenavir that inhibits pepsin and demonstrated the antireflux therapeutic potential of its prodrug fosamprenavir in a mouse model of laryngopharyngeal reflux. In this study, we assessed the capacity of amprenavir to protect against esophageal epithelial barrier disruption in vitro and related molecular events, E-cadherin cleavage, and matrix metalloproteinase induction, which are associated with GERD severity and esophageal cancer. Herein, weakly acidified pepsin (though not acid alone) caused cell dissociation accompanied by regulated intramembrane proteolysis of E-cadherin. Soluble E-cadherin responsive matrix metalloproteinases (MMPs) were transcriptionally upregulated 24 h post-treatment. Amprenavir, at serum concentrations achievable given the manufacturer-recommended dose of fosamprenavir, protected against pepsin-induced cell dissociation, E-cadherin cleavage, and MMP induction. These results support a potential therapeutic role for amprenavir in GERD recalcitrant to PPI therapy and for preventing GERD-associated neoplastic changes.

Keywords: Barrett’s esophagus; amprenavir; antireflux therapeutics; esophageal adenocarcinoma; fosamprenavir; gastroesophageal reflux disease; pepsin; protease inhibitors.

Figure 1

Amprenavir (10 µM) rescued cell dissociation caused by 1 mg/mL pepsin at pH4. The partial rescue was achieved by 1 µM APR. Acid alone did not cause cell dissociation. (A) Representative images of experiments performed in triplicate. Scale bars equal 100 µm. (B) Confluence as calculated using the FIJI plug-in PHANTAST (see Methods for details). Where significant, the following comparisons are shown: control vs. pH4, control vs. pepsin pH4, pepsin pH4 vs. +1 µM APR, and pepsin pH4 vs. +10 µM APR. *** = p < 0.001, **** = p < 0.0001.

Figure 2

Western blot (A) analyzed by densitometry (B) showed that acidified pepsin elicited E-cadherin cleavage via regulated intramembrane proteolysis (RIP), producing C-terminal intracellular fragments of 33 and 38 kDa. Acid alone did not cause E-cadherin RIP. 10 µM amprenavir partially rescued E-cadherin RIP, with increased full-length E-cadherin and decreased 33 and 38 kDa fragments compared to acidified pepsin, whereas 1 µM amprenavir resulted in a slight increase in full-length E-cadherin only. Dots represent samples for which no bands were detected. Where significant and where bands were detectable for analysis, the following comparisons are shown: control vs. pH4, control vs. pepsin pH4, pepsin pH4 vs. +1 µM APR, and pepsin pH4 vs. +10 µM APR. * = p < 0.05, ** = p < 0.01, **** = p < 0.0001.

Figure 3

Expression of MMPs following mock treatment, treatment with acidified pepsin ±1 or 10 µM amprenavir, or acid alone. Where significant, the following comparisons are shown: control vs. pH4, control vs. pepsin pH4, pepsin pH4 vs. +1 µM APR, and pepsin pH4 vs. +10 µM APR. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.