Abstract
Bile-containing gastro-duodenal reflux has been clinically considered an independent risk factor in hypopharyngeal carcinogenesis. We recently showed that the chronic effect of acidic bile, at pH 4.0, selectively induces NF-κB activation and accelerates the transcriptional levels of genes, linked to head and neck cancer, in normal hypopharyngeal epithelial cells. Here, we hypothesize that NF-κB inhibition is capable of preventing the acidic bile-induced and cancer-related mRNA phenotype, in treated normal human hypopharyngeal cells. In this setting we used BAY 11-7082, a specific and well documented pharmacologic inhibitor of NF-κB, and we observed that BAY 11-7082 effectively inhibits the acidic bile-induced gene expression profiling of the NF-κB signaling pathway (down-regulation of 72 out of 84 analyzed genes). NF-κB inhibition significantly prevents the acidic bile-induced transcriptional activation of NF-κB transcriptional factors, RELA (p65) and c-REL, as well as genes related to and commonly found in established HNSCC cell lines. These include anti-apoptotic bcl-2, oncogenic STAT3, EGFR, ∆Np63, TNF-α and WNT5A, as well as cytokines IL-1β and IL-6. Our findings are consistent with our hypothesis demonstrating that NF-κB inhibition effectively prevents the acidic bile-induced cancer-related mRNA phenotype in normal human hypopharyngeal epithelial cells supporting an understanding that NF-κB may be a critical link between acidic bile and early preneoplastic events in this setting.
Highlights
Many forms of Head and neck squamous cell carcinoma (HNSCC) have been attributed to known risk factors such as tobacco smoking and alcohol consumption [1,2,3], whereas extra-esophageal or laryngopharyngeal reflux disease (ERD or LPR) is considered to be an independent risk factor in laryngopharyngeal carcinogenesis [4,5,6]
Bile at molar concentrations previously reported in symptomatic gastro-esophageal reflux disease (GERD) patients [8] at pH ≤ 4.0 can induce significant activation of NF-κB and related genes associated with oncogenic function, in both cultured normal hypopharyngeal keratinocytes and premalignant lesions of exposed murine laryngopharyngeal mucosa www.impactjournals.com/oncotarget
To further analyze the effect of BAY 11-7082 on NFκB activation and bcl-2 expression, we performed a western blot analysis for phospho-NF-κB (p65 S356) (~65 kDa), phospho-inhibitor kappaB-a (p-IκB-α S32/S36) (~40 kDa) and bcl-2 (~28 kDa), using Histone 1 (~30 kDa) and β-actin (~37 kDa) for normalization of the expression, in nuclear and cytoplasmic protein fractions of the treated cells (Figure 2). (i) We observed an inhibition of NF-κB activation and bcl-2 cytoplasmic accumulation in both hypopharyngeal primary cells (HHPC) and hypopharyngeal keratinocytes (HHK) exposed to bile at pH 4.0 with BAY 11-7082, compared to cells exposed to bile at pH 4.0 without BAY 11-7082. This inhibition was evidenced by significantly decreased phospho-NF-κB nuclear levels in cells exposed to bile at pH 4.0 (Figure 2A-a, 2B-a), accompanied by reduced cytoplasmic p-IκB-α levels (Figure 2A-b, 2B-b) and cytoplasmic bcl-2 ratios (Figure 2A-c, 2B-c) (p < 0.05; by paired t-test; Graph Pad Prism 6.0). (ii) We found a decrease in NF-κB activation in HHPC exposed to acid plus BAY 11-7082, compared to those exposed to acid alone, without NF-κB inhibitor, demonstrating that NF-κB inhibitor suppressed NF-κB activation induced by low pH (Figure 2A-a, 2B-a). This event was evidenced by www.impactjournals.com/oncotarget a decrease of cytoplasmic p-IκB-α levels in cells exposed to acid alone (Figure 2A-b, 2B-b). (iii) On the other hand, we found minimal changes of nuclear NF-κB (Figure 2A-a, 2B-a) and cytoplasmic p-IκB-α (Figure 2Ab, 2B-b) levels in both HHPC and HHK treated with bile at pH 7.0 plus BAY 11-7082, compared to those exposed to bile at pH 7.0 without BAY 11-7082, again supporting our observations by IF assay
Summary
Many forms of Head and neck squamous cell carcinoma (HNSCC) have been attributed to known risk factors such as tobacco smoking and alcohol consumption [1,2,3], whereas extra-esophageal or laryngopharyngeal reflux disease (ERD or LPR) is considered to be an independent risk factor in laryngopharyngeal carcinogenesis [4,5,6]. In the initiation and progression of HNSCC, several oncogenic pathways have been identified These commonly include EGFR/Ras/RAF/MAPK, Akt/PI3K/ mTOR, ΙΚΚ/NF-κB, STAT3, and wnt/β-catenin [11,12,13,14,15,16,17,18,19,20]. 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 [21,22,23]. We hypothesize that NF-κB inhibition is capable of preventing the acidic bile-induced and cancer-related mRNA phenotype in treated normal hypopharyngeal cells, in vitro, further emphasizing the understanding that NFκB is a critical link between acidic bile and preneoplastic events
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