Pepsin enhances glycolysis to promote malignant transformation of vocal fold leukoplakia epithelial cells with dysplasia

Abstract

Purpose The mechanism underlying malignant transformation of vocal fold leukoplakia (VFL) and the precise role of the expression of pepsin in VFL remain unclear. This study aimed to investigate the effects of acidified pepsin on VFL epithelial cell growth and migration, and also identify pertinent molecular mechanisms. Methods Immunochemistry and Western blotting were performed to measure glucose transporter type 1 (GLUT1), monocarboxylate transporters 4 (MCT4), and Hexokinase-II (HK-II) expressions. Cell viability, cell cycle, apoptosis, and migration were investigated by CCK-8 assay, flow cytometry and Transwell chamber assay, respectively. Glycolysis-related contents were determined using the corresponding kits. Mitochondrial HK-II was photographed under a confocal microscope using Mito-Tracker Red. Results It was found: the expression of pepsin and proportion of pepsin+ cells in VFL increased with the increased dysplasia grade; acidified pepsin enhanced cell growth and migration capabilities of VFL epithelial cells, reduced mitochondrial respiratory chain complex I activity and oxidative phosphorylation, and enhanced aerobic glycolysis and GLUT1 expression in VFL epithelial cells; along with the transfection of GLUT1 overexpression plasmid, 18FFDG uptake, lactate secretion and growth and migration capabilities of VFL epithelial cell were increased; this effect was partially blocked by the glycolysis inhibitor 2-deoxy-glucose; acidified pepsin increased the expression of HK-II and enhanced its distribution in mitochondria of VFL epithelial cells. Conclusion It was concluded that acidified pepsin enhances VFL epithelial cell growth and migration abilities by reducing mitochondrial respiratory complex I activity and promoting metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis.

Inhaled fosamprenavir for laryngopharyngeal reflux: Toxicology and fluid dynamics modeling

“…It is therefore reasonable to assume that one or more nonacid constituents of refluxate are responsible for LPR symptoms. Among nonacid components of refluxate, the digestive enzyme pepsin is considered a predominant damaging agent, biomarker, and therapeutic target for reflux‐attributed diseases 5,26–35 . At the pH of the laryngopharynx (pH ~ 6.8), pepsin is enzymatically inactive, whereas studies in experimental models demonstrate that it is endocytosed and retained in acidic endosomes in which its enzymatic activity would be restored.…”

“…Among nonacid components of refluxate, the digestive enzyme pepsin is considered a predominant damaging agent, biomarker, and therapeutic target for reflux‐attributed diseases. 5 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 At the pH of the laryngopharynx (pH ~ 6.8), pepsin is enzymatically inactive, whereas studies in experimental models demonstrate that it is endocytosed and retained in acidic endosomes in which its enzymatic activity would be restored. Exposure to pepsin leads to inflammatory and carcinogenic alterations irrespective of pH in vitro and in vivo including altered transcriptomic profiles; promotion of apoptotic resistance, cell migration, anchorage‐independent growth, and glycolysis; and development of tumors in a hamster cheek model.…”

“…Exposure to pepsin leads to inflammatory and carcinogenic alterations irrespective of pH in vitro and in vivo including altered transcriptomic profiles; promotion of apoptotic resistance, cell migration, anchorage‐independent growth, and glycolysis; and development of tumors in a hamster cheek model. 31 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 …”

“…Exposure to pepsin leads to inflammatory and carcinogenic alterations irrespective of pH in vitro and in vivo including altered transcriptomic profiles; promotion of apoptotic resistance, cell migration, anchorage-independent growth, and glycolysis; and development of tumors in a hamster cheek model. 31,[33][34][35][36][37][38][39][40] We recently identified the protease inhibitor fosamprenavir (FOS; prodrug of amprenavir, APR), an FDA-approved therapy for HIV/AIDS, as a candidate therapy for LPR. FOS binds and inhibits pepsin in the low micromolar range and oral gavage of FOS at the manufacturer-recommended dose for HIV prevented inflammatory damage in an LPR mouse model.…”

Amprenavir inhibits pepsin‐mediated laryngeal epithelial disruption and E‐cadherin cleavage in vitro

“…Pepsin is frequently detected in airway tissue and secretions from LPR patients yet absent in MII‐pH‐confirmed reflux‐free subjects 28–31 . Its abundance and correlation with cancer‐associated changes such as increased interleukin‐8 and DNA damage in dysplastic or tumor airway tissue support its contribution to the elevated risk of laryngeal cancer among LPR patients 5,32–37 . In experimental models, pepsin leads to inflammatory and carcinogenic changes irrespective of pH in vitro and in vivo including altered transcriptomic profiles; promotion of apoptotic resistance, cell migration, anchorage‐independent growth and glycolysis; and development of tumors in a hamster cheek model 27,33,35–41 .…”