Poster (R5)- #STPE50 - KEYWORDS: GERD, gastroesophageal reflux, in vivo, Bile reflux, carcinogenesis, hypopharyngeal cancer, curcumin, cancer prevention

The in vivo preventive and therapeutic properties of curcumin in bile reflux‐related oncogenesis of the hypopharynx

Sotirios G. Doukas, MD1 Panagiotis G. Doukas, MD,2 Clarence T. Sasaki, MD2, Dimitra P. Vageli, PhD2
1 Department of Medicine, Rutgers/Saint Peter’s University Hospital, New Brunswick, New Jersey; 2 The Yale Larynx laboratory, Department of Surgery, Yale School of Medicine, New Haven, CT , USA

ABSTRACT:
Supra-esophageal reflux has been causally implicated epidemiologically in hypopharyngeal cancer.1 Almost 50-86% of GERD (gastroesophageal reflux disease) patients have been shown to present with mixed gastric and duodenal fluids in esophageal refluxate suggesting that enterogastric reflux may be much more prevalent than previously appreciated.2,3 Although reflux episodes often occur at pH 4.0, they can also occur at weakly acidic pH 5.5-6.0.4,5 The carcinogenic effect of bile at a strongly acidic pH 3.0, was recently documented in vivo.6,7 Because esophageal refluxate often occurs at pH >4.0, we aimed to determine whether weakly acidic bile is also carcinogenic in vivo.We used 32 wild-type mice C57B16J, and performed topical application of conjugated primary bile acids with or without unconjugated secondary bile acid, deoxycholic acid (DCA), at pH 5.5 and controls, to hypopharyngeal mucosa (HM) twice per day, for 15 weeks. We performed histologic staining (hematoxylin and eosin, H&E) and histologic evaluation based on previously established criteria; immunohistochemical analysis for NF-kB, p53, Ki67, CK14, E-Cadherin, H2AX, and DNA/RNA oxidative damage markers, and mRNA and miRNA analysis for Bcl2, Il6, Tnf, Egfr, Wnt5a, Rela, Stat3, miR-21, -155, -192, -34a, -375, and -451a. Chronic exposure of hypopharyngeal mucosa to weakly acidic bile, promotes premalignant lesions with microinvasion (Figure 1A), preceded by significant DNA/RNA oxidative damage, γH2AX (double strand breaks), NF-κB and p53 expression, overexpression of Bcl-2, and elevated Tnf, Il6 mRNAs, compared to controls (Figure 1B,C). Weakly acidic bile upregulates the “oncomirs”, miR-21 and -155. The presence of DCA promotes Egfr, Wnt5a, Rela overexpression, and a significant downregulation of “tumor suppressor” miR-451a (Figure 1C,D). Weakly acidic pH increases the risk of bile-related hypopharyngeal neoplasia. The oncogenic properties of biliary esophageal reflux on supra-esophageal epithelium may be not fully modified when antacid therapy is applied. We believe that due to bile content, alternative therapeutic strategies using specific inhibitors of relevant molecular pathways or receptors may be considered in patients with refractory GERD. 1Parsel SM, et al. Clin Gastroenterol Hepatol. 2019; 2Covington MF, et al. J Nucl Med Technol. 2014; 3McQuaid KR, et al. Aliment Pharmacol Ther. 2011; 4Hemmink GJ, et al. Am J Gastroenterol. 2008; 5Kauer WK, Stein HJ. Chest Surg Clin N Am. 2002; 6Vageli DP, Prasad ML, Sasaki CT. Oncotarget 2016; 7Sasaki CT, Doukas SG, Costa, J, Vageli, DP. Cancers 2020.