Pepsin promotes proliferation of laryngeal and pharyngeal epithelial cells

Abstract

Objective/Hypothesis Laryngopharyngeal reflux (LPR) is thought to be a significant risk factor for laryngeal squamous cell carcinoma (SCC), but causality has never been proven. It is accepted that chronic reflux into the esophagus can induce metaplastic changes in esophageal mucosa with subsequent increased risk of esophageal adenocarcinoma, but no similar associations have been established for LPR and laryngopharyngeal SCC. The objective of this study was to test the hypothesis that reflux of pepsin into the laryngopharynx can promote carcinogenesis. Study Design Translational research study Methods Normal human laryngeal primary epithelial cell cultures and hypopharyngeal FaDu SCC cells were exposed to human pepsin and analyzed by Human Cancer PathwayFinder and miRNA Superarrays, flow cytometry and Western blot to determine the effect of pepsin on carcinogenesis. Laryngeal biopsy specimens, taken from cancer patients and normal control subjects, were analyzed for the presence of pepsin by Western blot. Results Microarray analysis demonstrated that pepsin significantly altered the expression of 27 genes implicated in carcinogenesis and also affected the expression of 22 microRNAs known to be altered in human head and neck cancers. Pepsin increased proliferation in both FaDu SCC cells and cultured normal laryngeal epithelial primary cells by increasing S phase distribution on flow cytometry analysis in a time and dose dependent manner. Furthermore, pepsin was detected in 60% (3/5) human laryngeal cancer biopsies, absent in all (0/5) normal control specimens. Conclusion These data support a role for refluxed pepsin in the promotion of epithelial proliferation and carcinogenesis of the larynx and pharynx.

The Role of Pepsin in LPR: Will It Change Our Diagnostic and Therapeutic Approach to the Disease?

“…Using an in vivo hamster buccal pouch model of squamous cell carcinoma (SCC), our group found a significant increase in tumor volume in hamster cheek pouches exposed to pepsin (0.1 mg/ml; pH 2) plus a known carcinogen-7, 1 dimethylbenzanthracene (DMBA)-compared to exposure to DMBA alone, suggesting that pepsin in acid reflux does promote tumorigenesis [54]. In addition, our group has reported that exposure of hypopharyngeal FaDu cells and normal human primary epithelial cells to pepsin (0.1 mg/ ml; pH 7) causes a significant change in the expression of 27 genes implicated in carcinogenesis and of 22 microRNAs specifically known to be altered in head and neck SCC [6]. Pepsin increased proliferation in both FaDu SCC cells and normal laryngeal primary epithelial cells by significantly increasing the percentage of cells in S phase in a time-and dose-dependent manner.…”

“…Real-time (RT)-polymerase chain reaction (PCR) and Western blot analysis has been used to confirm that the laryngopharynx does not produce pepsinogen A or pepsin in LPR patients or healthy controls [6,39]. Thus, pepsin in the laryngopharynx is a specific marker for LPR [6,16].…”

“…While serum pepsinogen may be present in the airway tissues of patients with gastritis and pepsinogen has been reported to be produced by metaplastic tissue of the esophagus, production of mature pepsin protein has only been reported in the stomach [34][35][36][37][38]. Real-time (RT)-polymerase chain reaction (PCR) and Western blot analysis has been used to confirm that the laryngopharynx does not produce pepsinogen A or pepsin in LPR patients or healthy controls [6,39]. Thus, pepsin in the laryngopharynx is a specific marker for LPR [6,16].…”

“…Given pepsin's role in nonacidic LPR, it has been proposed as a novel therapeutic target, especially for patients experiencing refractory symptoms on PPIs [5,6,[45][46][47]. Approximately $26 billion/year is currently being spent on PPIs for the treatment of LPR, despite their poor efficacy for this patient population [12].…”

“…LPR affects children and adults equally, and the clinical spectrum of this disease is extensive [1][2][3]. Unlike patients with gastroesophageal reflux (GER), which is limited to the esophagus, many LPR patients do not experience heartburn but present with symptoms due to chronic laryngeal irritation and inflammation such as chronic cough, throat clearing, post nasal drip, hoarseness or dysphonia, globus sensation, dysphagia, and dyspnea [1,4] and significant evidence exists that chronic LPR contributes to lifethreatening illness such as laryngeal cancer [5][6][7]. El-Serag et al [8] showed the annual average rate of increase of reflux disease since 1976 was 4 %.…”