Secondary Bile Acids Contribute to Intestinal Epithelial Cell Injury via Inhibition of Cell Migration

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Surgical Forum Abstracts

Silencing Gastrin-Releasing Peptide Receptor Suppresses Key Regulators of Aerobic Glycolysis in Neuroblastoma Cells Carmelle Romain, MD, SunPhil Choi, PhD, Jingbo Qiao, PhD, Dai H Chung, MD, FACS Vanderbilt University Medical Center, Nashville, TN INTRODUCTION: Under normoxic conditions, cancer cells use aerobic glycolysis as opposed to glucose oxidation for energy production. This altered metabolic phenotype correlates with poor outcomes in neuroblastoma. Hypoxia-inducible factor-1 alpha (HIF-1a) and pyruvate dehydrogenase kinase 4 (PDK4) positively regulate aerobic glycolysis, while pyruvate dehydrogenase phosphatase 2 (PDP2) promotes glucose oxidation. We showed that gastrin-releasing peptide receptor (GRPR) signaling promotes aggressive phenotype in neuroblastoma. Here, we sought to determine whether GRPR regulates the glucose metabolic pathway and its effect on neuroblastoma tumorigenesis. METHODS: Gene expression for glucose metabolic pathway was performed after silencing GRPR. Expression of HIF-1a and PDK4 were assessed after cobalt chloride (CoCl2) treatment (100 mM), a chemical hypoxia mimetic, with or without silencing PDK4 (siPDK4). ELISA was used to measure VEGF secretion. Dichloroacetate (DCA), a glycolysis inhibitor, was used to determine its effect on PDC, an enzyme that positively regulates aerobic glycolysis when phosphorylated. Cell viability was measured after treatment with DCA. RESULTS: Silencing GRPR decreased the expression of HIF-1a and VEGF when compared to controls. PDK4 expression was decreased while PDP2 increased after silencing GRPR. Hypoxiainduced increases of VEGF secretion, HIF-1a and PDK4 expression were reversed after silencing PDK4. DCA treatment inhibited PDC phosphorylation and decreased cell proliferation at 72 h. CONCLUSIONS: Our findings demonstrate that GRPR signaling is critical for tumor glucose metabolism by regulating PDK4 and PDP2. PDK4 may control glucose metabolism via regulation of HIF-1a. DCA could provide a novel therapy in the treatment of aggressive neuroblastoma driven, in part, by aerobic glycolysis. Secondary Bile Acids Contribute to Intestinal Epithelial Cell Injury via Inhibition of Cell Migration Stephanie C Papillon, MD, Avafia Y Dossa, MD, MS, Anne S Roberts, MD, Mark R Frey, PhD, Henri R Ford, MD, FACS, Christopher P Gayer, MD, PhD Children’s Hospital Los Angeles, Los Angeles, CA INTRODUCTION: Necrotizing enterocolitis remains a significant gastrointestinal emergency in premature neonates. Studies have implicated secondary bile acids, produced by modification of bile acids by intestinal bacteria, in disease pathogenesis. We hypothesized that secondary bile acids may promote mucosal injury by inhibiting epithelial cell migration. METHODS: After 24 hours of serum-starvation, intestinal epithelial cell (IEC-6) monolayers were subjected to a modified wound

J Am Coll Surg

restitution assay in the presence of bile acids, epidermal growth factor (EGF), the EGF receptor inhibitor AG1478, or a combination of these. Pictures taken at 0 and 6 hours were compared. RESULTS: Both unconjugated secondary bile acids, deoxycholic acid (DCA) and lithocolic acid (LCA), significantly reduced epithelial cell migration in a dose-dependent manner (11% and 30% below control, respectively). The most effective dose was the highest non-toxic dose as measured via an MTS-based viability assay. LCA reduced migration as much as EGFR inhibition with AG1478. While conjugated forms of DCA did not alter migration, taurine-conjugated LCA significantly reduced migration (8% below control), although much less than the unconjugated LCA. DCA-induced inhibition of migration was overcome by adding EGF. However, EGF treatment did not reverse the inhibitory effects of LCA on epithelial cell migration. CONCLUSIONS: Our data demonstrate that secondary bile acids impair epithelial cell migration, with unconjugated metabolites inhibiting migration more than conjugated forms. LCA may act downstream of the EGF receptor pathway since addition of EGF did not reverse its effect on epithelial cell migration. These findings suggest a mechanism by which the intestinal microbiota can lead to gastrointestinal disease. Human Amniotic Fluid-Derived Multipotent Stromal Cells Enhance Decellularized Diaphragm Scaffold Regeneration and Function in a Rodent Model of Congenital Diaphragmatic Hernia George P Liao, MD, Kinga Vojnits, PhD, Yohan Choi, PhD, Kevin R Aroom, MS, Robert A Hetz, MD, Hasen Xue, MD, Fabio Triolo, PhD, Yong Li, MD, PhD, Kevin P Lally, MD, FACS, Charles S Cox, MD, FACS University of Texas Health Science Center at Houston, Houston, TX INTRODUCTION: Tissue engineering for congenital diaphragmatic hernias (CDH) repair seeks to improve biomechanical compatibility while reducing device failure. Our lab explored using human amniotic fluid-derived multipotent stromal cells (hAFMSC) to enhance decellularized diaphragm scaffolds in a preclinical rodent model for potential translational repair of CDH diagnosed in utero. METHODS: AFMSCs were isolated via amniocentesis from pregnant women at our institution undergoing therapeutic amnioreduction and introduced onto decellularized rat diaphragms and implanted in rats with surgically created left sided diaphragmatic hernias (n¼6). Control rats (n¼5) received decellularized diaphragms alone. At 4 months post-implantation, diaphragms were explanted and subjected to physiologic testing and viable tissue (n¼2-6) from each group underwent immunohistochemical analysis with the native contralateral hemidiaphragm as control. RESULTS: Single pulse as well as train of pulses stimulation of explanted hemidiaphragms containing scaffolds treated with hAFMSCs generated 433% and 364% respectively of the