Temporal characteristics of NF-κB inhibition in blocking bile-induced oncogenic molecular events in hypopharyngeal cells

// Panagiotis G. Doukas 1 , Dimitra P. Vageli 1 , Sotirios G. Doukas 1 and Clarence T. Sasaki 1 1 The Yale Larynx Laboratory, Department of Surgery, Yale School of Medicine, New Haven, CT, USA Correspondence to: Clarence T. Sasaki, email: clarence.sasaki@yale.edu Keywords: NF-κB inhibition; STAT3; miR-21; bile reflux; hypopharyngeal cancer Received: March 12, 2019     Accepted: April 21, 2019     Published: May 21, 2019 ABSTRACT Biliary esophageal reflux at acidic pH is considered a risk factor in laryngopharyngeal cancer. We previously showed the key role NF- κ B in mediating acidic bile-induced pre-neoplastic events in hypopharyngeal cells, and that co-administration of specific NF- κ B inhibitor, BAY 11-7082, together with acidic bile, can effectively prevent its related oncogenic molecular effects. We hypothesize that the addition of BAY 11-7082 (10μM) either before or after application of acidic bile (400μM conjugated bile acids; pH 4.0), is capable of comparably blocking acidic bile-induced oncogenic molecular phenotypes in murine hypopharyngeal primary cells. We performed immunofluorescence, luciferase assay, western blot and qPCR analysis, demonstrating that 15-min of pre- or post-application of BAY 11-7082 effectively inhibits acidic bile-induced NF- κ B activation, transcriptional activation of RELA(p65), STAT3, EGFR, IL-6, bcl-2, W NT5A , “upregulation” of “oncomirs” miR-21, miR-155, miR-192 and “downregulation” of “tumor suppressor” miR-34a, miR-375, miR-451a. Our observations support the understanding that acidic bile-induced deregulation of anti-apoptotic or oncogenic factors, bcl-2 , STAT3, EGFR, IL-6, WNT5A , miR-21, miR-155, miR-375, is highly NF- κ B-dependent, showing that even post-application of inhibitor can suppress their deregulation. In conclusion, application of specific NF-κB inhibitor, has the capability of adequately blocking the early oncogenic molecular events produced by acidic bile whether it is applied pre or post exposure. In addition to therapeutic implications these findings provide a window of observation into the complex kinetics characterizing the mechanistic link between acidic bile and early neoplasia. Although BAY 11-7082 itself may not be suitable for clinical use, the application of other NF-κB inhibitors merits exploration.