Pepsin promotes IL-8 signaling-induced epithelial-mesenchymal transition in laryngeal carcinoma

Abstract

Background: Laryngopharyngeal reflux (LPR), with its increasing morbidity, is attracting considerable attention. In recent years, the causal role between LPR and laryngeal carcinoma has been debated. The main harmful component of LPR is pepsin, which has been shown to induce mucosal inflammation by damaging the mucous membrane. Thus, pepsin is linked to an increased risk of laryngeal carcinoma, although the potential mechanism remains largely unknown.

Methods: The human laryngeal carcinoma cell lines Hep-2 and Tu212 were exposed to different pepsin concentrations and the morphology, proliferation, migration, secretion of inflammatory cytokines, and epithelial-mesenchymal transition (EMT) of the cells were assessed. To evaluate whether interleukin-8 (IL-8) had a causal relationship with pepsin and EMT, an IL-8 inhibitor was used to suppress IL-8 secretion during pepsin exposure and the expression of EMT markers, cell proliferation, and migration were analyzed.

Results: Pepsin promoted proliferation, colony formation, migration, and IL-8 secretion of Hep-2 and Tu212 cells in vitro. Furthermore, increased pepsin concentrations changed the morphology of Hep-2 and Tu212 cells; levels of the epithelial marker E-cadherin were reduced and those of mesenchymal markers vimentin and β-catenin and the transcription factors snail and slug were elevated. A similar effect was observed in laryngeal carcinoma tissues using immunohistochemistry. IL-8 level was reduced and EMT was restored when pepsin was inhibited by pepstatin. EMT was weakened after exposure to the IL-8 inhibitor, with significant reduction in pepsin-induced cell proliferation and migration.

Conclusions: Pepsin may induce EMT in laryngeal carcinoma through the IL-8 signaling pathway, which indicates that it has potential role in enhancing cell proliferation and metastasis of laryngeal carcinoma.

Keywords: Laryngeal carcinoma; Laryngopharyngeal reflux; Pepsin.

Fig. 1

Proliferative ability of Hep-2 and Tu212 cells treated with different concentrations of pepsin. a Effect of different concentrations of pepsin on proliferation of Hep-2 and Tu212 cells measured using Edu assays (magnification, ×200). b Representative images of the colony formation assay of Hep-2 and Tu212 cells at different pepsin concentrations. c Representative histograms of cell cycle assays showing the percentage of Hep-2 and Tu212 cells in S phase at different pepsin concentrations. d Migratory properties of Hep-2 and Tu212 cells exposed to different concentrations of pepsin were analyzed using transwell migration assays (magnification, ×200). *P < 0.05 compared to that of the controls

Fig. 2

Expression of IL-8 after pepsin exposure. a Change in inflammatory cytokine expression of Hep-2 and Tu212 cells exposed to different concentrations of pepsin measured using the CBA assay. Production of IL-8 and IL-6 by Hep-2 and Tu212 cells at different pepsin concentrations. b Change in inflammatory cytokine expression of Hep-2 and Tu212 cells exposed to pepsin with/without pepstatin measured using CBA assays. Production of IL-8 and IL-6 by Hep-2 and Tu212 cells at different concentrations of pepsin. c Photomicrographs representative of the immunohistochemical analyses of pepsin and IL-8 in tissue specimens from three patients with laryngeal carcinoma (magnification, ×400). *P < 0.05 compared to that of the controls

Fig. 3

Expression of epithelial and mesenchymal markers in Hep-2 and Tu212 cells treated with pepsin. a Morphology of Hep-2 and Tu212 cells exposed to different pepsin concentrations is shown using phase contrast microscopy. b Expression levels of E-cadherin, vimentin, and β-catenin in Hep-2 and Tu212 cells exposed to different concentrations of pepsin analyzed using quantitative real-time PCR. c Effect of different pepsin concentrations on the expression of E-cadherin and vimentin in Hep-2 and Tu212 cells immunostained and analyzed using confocal microscopy (magnification, ×200). d Expression of E-cadherin, vimentin, β-catenin, snail, and slug in Hep-2 and Tu212 cells exposed to different concentrations of pepsin analyzed using western blotting. e Expression of E-cadherin, vimentin, β-catenin, snail, and slug in Hep-2 and Tu212 cells exposed to pepsin with/without pepstatin analyzed using western blotting. f Representative photomicrographs illustrating the immunohistochemical analyses for pepsin, E-cadherin, vimentin, and β-catenin in tissue specimens from two patients with laryngeal carcinoma (magnification, ×400). *P < 0.05 compared to that of the controls

Fig. 4

In vitro Hep-2 and Tu212 cells exposed to pepsin after the blockage of IL8 receptors. a Western blot analysis of E-cadherin, vimentin, β-catenin, snail, and slug expression in Hep-2 and Tu212 cells exposed to pepsin with or without SB225002 treatment to inhibit the IL-8 receptor CXCR2. b Effect of pepsin on Hep-2 and Tu212 cell proliferation with or without SB225002 treatment to inhibit the IL-8 receptor CXCR2. Cell proliferation was measured using Edu assays (magnification, ×100). c Representative histograms of cell cycle assays showing the percentage of pepsin-treated Hep-2 and Tu212 cells in S phase with or without SB225002 treatment to inhibit the IL-8 receptor CXCR2. d Migratory properties of Hep-2 and Tu212 cells exposed to pepsin with or without inhibition of the IL-8 receptor CXCR2 by SB225002 treatment analyzed using transwell migration assays, with the number of migrated Hep-2 and Tu212 cells shown (magnification, ×200). *P < 0.05 compared to that of the controls

Fig. 5

Pepsin may induce EMT in laryngeal carcinoma, underlining its potential role in enhancing laryngeal carcinoma proliferation and metastasis associated with IL-8 secretion