Abstract
BackgroundExtra-esophageal carcinogenesis has been widely discussed in relation to the chronic effects of laryngopharyngeal reflux and most prominently with pepsin historically central to this discussion. With refluxate known to include gastric (pepsin) and duodenal (bile) fluids, we recently demonstrated the mechanistic role of NF-κB in mediating the preneoplastic effects of acidic-bile. However, the role of pepsin in promoting hypopharyngeal premalignant events remains historically unclear. Here, we investigate the in vitro effect of acidic-pepsin on the NF-κB oncogenic pathway to better define its potential role in hypopharyngeal neoplasia.MethodsHuman hypopharyngeal primary cells (HHPC) and keratinocytes (HHK) were repetitively exposed to physiologic pepsin concentrations (0.1 mg/ml) at pH 4.0, 5.0 and 7.0. Cellular localization of phospho-NF-κB and bcl-2 was determined using immunofluorescence and western blotting. NF-κB transcriptional activity was tested by luc reporter and qPCR. Analysis of DNA content of pepsin treated HHK and HHPC was performed using Fluorescence-activated-cell sorting assay. To explore a possible dose related effect, pepsin concentration was reduced from 0.1 to 0.05 and 0.01 mg/ml.ResultsAt physiologic concentration, acidic-pepsin (0.1 mg/ml at pH 4.0) is lethal to most normal hypopharyngeal cells. However, in surviving cells, no NF-κB transcriptional activity is noted. Acidic-pepsin fails to activate the NF-κB or bcl-2, TNF-α, EGFR, STAT3, and wnt5α but increases the Tp53 mRNAs, in both HHPC and HHK. Weakly acidic-pepsin (pH 5.0) and neutral-pepsin (pH 7.0) induce mild activation of NF-κB with increase in TNF-α mRNAs, without oncogenic transcriptional activity. Lower concentrations of pepsin at varying pH do not produce NF-κB activity or transcriptional activation of the analyzed genes.ConclusionOur findings in vitro do not support the role of acidic-pepsin in NF-κB related hypopharyngeal carcinogenesis.
Highlights
The American Cancer Society estimates approximately 3,000 cancers will start in the hypopharynx in 2016 but only 53% of cases even at early stages will survive 5-years [1,2]
The purpose of this study is to clarify whether constitutive stimulation of human hypopharyngeal primary cells (HHPC) and immortalized human hypopharyngeal keratinocytes (HHK) with acidic, weakly acidic or neutral pepsin is capable of inducing transcriptional activation of anti-apoptotic genes, including bcl-2, cell signaling TNF-α, oncogenic EGFR, STAT3, wnt5α, Tp63 or altering the expression of cell cycle control-related Tp53 linked to head and neck squamous cell carcinoma (HNSCC) [18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]
Both HHK and HHPC repetitively exposed to 0.1 mg/ml of acidic-pepsin demonstrated cytoplasmic p-NF-κB (p-p65 S529) staining with a few sporadic cells that were positive for p-p65 nuclear staining (Fig 1A and 1B)
Summary
The American Cancer Society estimates approximately 3,000 cancers will start in the hypopharynx in 2016 but only 53% of cases even at early stages will survive 5-years [1,2]. Esophageal and llaryngopharyngeal reflux disease (ERD or LPR), are considered potential risk factors in hypopharyngeal carcinogenesis [5,6,7]. We recently provided evidence in vitro and in vivo of the potential role of gastroduodenal fluid and of bile at acidic pH ( 4.0) in hypopharyngeal neoplasia, mediated by the NF-κB activated pathway [10,11]. Extra-esophageal carcinogenesis has been widely discussed in relation to the chronic effects of laryngopharyngeal reflux and most prominently with pepsin historically central to this discussion. With refluxate known to include gastric (pepsin) and duodenal (bile) fluids, we recently demonstrated the mechanistic role of NF-κB in mediating the preneoplastic effects of acidic-bile. We investigate the in vitro effect of acidic-pepsin on the NF-κB oncogenic pathway to better define its potential role in hypopharyngeal neoplasia
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