The Role of PARP-1 and NF-κB in Bile-Induced DNA Damage and Oncogenic Profile in Hypopharyngeal Cells

Objectives/Hypothesis: We recently documented that acidic bile, a gastroesophageal reflux content, can cause invasive hypopharyngeal squamous cell carcinoma, by inducing widespread DNA damage and promoting nuclear factor kappa B (NF-kappa B)-related oncogenic molecular events. Poly or adenosine diphosphate (ADP)-ribose polymerase-1 (PARP-1), a sensitive sensor of DNA damage, may interact with NF-kappa B. We hypothesized that PARP-1 is activated in hypopharyngeal cells (HCs) with marked DNA damage caused by acidic bile, hence there is an association between PARP-1 and NF-kappa B activation or its related oncogenic profile, in this process. Study Design: In vitro study. Methods: We targeted PARP-1 and NF-kappa B(p65), using pharmacologic inhibitors, 1.0 mu M Rucaparib (AG014699) and 10 mu M BAY 11-7082 {3-[4=methylphenyl)sulfonyl]-(2E)-propenenitrile}, respectively, or silencing their gene expression (siRNAs) and used immunofluorescence, luciferase, cell viability, direct enzyme-linked immunosorbent assays, and qPCR analysis to detect the effect of targeting PARP-1 or NF-kappa B in acidic bile-induced DNA damage, PARP-1, p-NF-kappa B, and B-cell lymphoma 2 (Bcl-2) expression, as well as NF-kappa B transcriptional activity, cell survival, and mRNA oncogenic phenotype in HCs. Results: We showed that (i) PARP-1 is overexpressed by acidic bile, (ii) targeting NF-kappa B adequately prevents the acidic bile-induced DNA double-strand breaks (DSBs) by gamma H2A histone family member X (gamma H2AX), oxidative DNA/RNA damage, PARP-1 overexpression, anti-apoptotic mRNA phenotype, and cell survival, whereas (iii) targeting PARP-1 preserves elevated DNA damage, NF-kappa B activation, and anti-apoptotic phenotype. Conclusion: We document for the first time that the activation of PARP-1 is an early event during bile reflux-related head and neck carcinogenesis and that NF-kappa B can mediate DNA damage and PARP-1 activation. Our data encourage further investigation into how acidic bile-induced activated NF-kappa B mediates DNA damage in hypopharyngeal carcinogenesis.