The In Vitro Effect of Acidic-Pepsin on Nuclear Factor KappaB Activation and Its Related Oncogenic Effect on Normal Human Hypopharyngeal Cells

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.

Pepsin promotes laryngopharyngeal neoplasia by modulating signaling pathways to induce cell proliferation

“…In an in vitro model of gastroduodenal reflux model, Sasaki et al [26] observed Bcl-2 overexpression and significant transcriptional deregulation of NF-κB-related genes with oncogenic function in hypopharyngeal cancer cells treated with acid/ bile. In another in vitro study performed by Sasaki el al [27], weakly acidic-pepsin (pH 5.0) and neutral-pepsin (pH 7.0) were found could induce mild activation of NF-κB with increase in TNF-α mRNAs in human hypopharyngeal primary cells, that is in accordance with our study, but no oncogenic transcriptional activity was detected in their study. This could be explained that the mild increase of NF-κB activity may be related to stress reaction [27] and in vitro cellular study could not mimic the dynamic events in tissue response to selected ranges of acidified pepsin.…”

Inhibition of NF-κB prevents the acidic bile-induced oncogenic mRNA phenotype, in human hypopharyngeal cells

“…Prior findings from our in vitro and in vivo models demonstrated that acidic bile can induce NF-κB activation and accelerate the transcriptional levels of genes related to oncogenic function [ 21 , 22 ]. We previously have shown that bile and acid in combination contributed significantly to NF-κB activation and bcl-2 overexpression in vitro , in treated normal hypopharyngeal cells, and in vivo , in murine hypopharyngeal mucosa, relative to acid alone or other factors such as topical glucose or pepsin [ 21 – 23 ]. We showed in vivo that this combination induced premalignant hypopharyngeal lesions, exhibiting increased cell proliferation rates and transcriptional activation of genes with anti-apoptotic or oncogenic function, such as EGFR, STAT3, TNF-α, wnt5A.…”

“…These commonly include EGFR/Ras/RAF/MAPK, Akt/PI3K/mTOR, ΙΚΚ/NF-κB, STAT3, and wnt/β-catenin [ 11 – 20 ]. In this setting, our recent in vitro and in vivo explorations demonstrate that extra-esophageal reflux may play a role in laryngopharyngeal carcinogenesis, mediated by NF-κB [ 21 – 23 ]. Recently published data demonstrate that bile, particularly at pH ≤ 4.0 is capable of inducing NF-κB activation and oncogenic mRNA phenotype [ 21 , 22 ].…”

The diagnostic value of pepsin detection in saliva for gastro-esophageal reflux disease: a preliminary study from China

“…GERD is a common but often misdiagnosed disease in clinical practice. Studies [ 21 – 28 ] showed that GERD was the underlying factor of many pulmonary and otolaryngology diseases and conditions, such as asthma, chronic cough, pneumonia, laryngitis, pharyngitis, hoarseness, and even malignant tumor of head and neck. Because of the nonspecific symptoms of reflux disease, the definitive diagnosis of GERD is still challenging.…”