Reflux induces DNA strand breaks and expression changes of MMP1+9+14 in a human miniorgan culture model.
Gastroesophageal reflux disease has been implicated in the pathogenesis of adenocarcinoma of the oesophagus. The same applies to laryngopharyngeal reflux (LPR) and squamous cell cancer of the head and neck, but so far, this link has not been proven. The impact of low pH and bile acids has not been studied extensively in cells other than oesophageal cancer cell lines and tissue. The aims of this study were to investigate the pathogenic potential of reflux and its single components on the mucosa of the upper respiratory tract. We measured DNA stability in human miniorgan cultures (MOCs) and primary epithelial cell cultures (EpCs) in response to reflux by the alkaline comet assay. As matrix metalloproteinases (MMPs) are involved in extracellular matrix remodelling processes and may contribute to cancer progression, we studied the expression of MMP1, -9, and -14 in MOCs, EpC, UM-SCC-22B, and FADUDD. DNA strand breaks (DNA-SBs) increased significantly at low pH and after incubation with human or artificial gastric juice. Single incubation with glycochenodeoxycholic acid also showed a significant increase in DNA-SBs. In epithelial cell cultures, human gastric juice increased the number of DNA-SBs at pH 4.5 and 5.5. Artificial gastric juice significantly up regulated the gene expression of MMP9. Western blot analysis confirmed the results of gene expression analysis, but the up regulation of MMP1, -9, and -14 was donor-specific. Reflux has the ability to promote genomic instability and may contribute to micro environmental changes suitable for the initiation of malignancy. Further functional gene analysis may elucidate the role of laryngopharyngeal reflux in the development of head neck squamous cell carcinoma (HNSCC).
Department of Otolaryngology, Head and Neck Surgery, Martin-Luther-University Halle-Wittenberg, D-06120 Halle, Germany. Electronic address: annett.sandner@uk-halle.de.
Annett
A
Sandner
Juliane
J
Illert
Sabine
S
Koitzsch
Susanne
S
Unverzagt
Ilona
I
Schön
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000502:physiology; D004847:Epithelial Cells / Q000378:metabolism; D004947:Esophagus / Q000378:metabolism; D005764:Gastroesophageal Reflux / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D005796:Genes / Q000502:physiology; D006258:Head and Neck Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000378:metabolism*; D053511:Matrix Metalloproteinase 14 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D000077195:Squamous Cell Carcinoma of Head and Neck; D014407:Tumor Cells, Cultured
D004247:DNA; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9; C522360:MMP1 protein, human; D020781:Matrix Metalloproteinase 1; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14
AECGM; AcOH; BA; BCA; BE; Barrett oesophagus; CK 19; Comet assay; DNA; DNA strand breaks; DNA-SBs; DNA-strand breaks; ECM; EpC; Expression changes of MMP1, 9, 14; FCS; GAPDH; GCDA; GJ; HCl; HE; HNSCC; HRP; Human miniorgan culture model; LPR; Laryngopharyngeal reflux; MEOH; MMPs; MOC; NF-κB; OTM; Olive tail moment; P; PBS; ROS; SBTI; SCC; SDS; T; UM; University of Michigan; acetic acid; airway epithelial cell growth medium; bicinchoninic acid; bile acid; cytokeratin 19; deoxyribonucleic acid; epithelial cell culture; extracellular matrix; foetal calf serum; gastric juice; glycerol phosphate dehydrogenase; glycochenodeoxycholic acid; head and neck squamous cell carcinoma; heamatoxylin eosin; heat inactivated pepsin; horseradish peroxidase; hydrochloric acid; iP; laryngopharyngeal reflux; matrix metalloproteinases; methanol; miniorgan culture; nuclear factor kappa B; pepsin; phosphate buffered saline; reactive oxygen species; sodium dodecyl sulphate; soy bean trypsin inhibitor; squamous cell carcinoma; trypsin