Background: Mechanisms by which alcohol increases the risk of esophageal squamous cell carcinoma remain undefined. Human esophageal myofibroblasts (HEMFs) subjacent to the squamous epithelium are exposed directly to these agents via epithelial barrier defects and indirectly via factors derived from the exposed epithelium. Our aim was to investigate the cellular biology of HEMFs and HEMF-esophageal epithelial cell interactions in response to alcohol and its toxic metabolite acetaldehyde.
Methods: An immortalized HEMF and a human esophageal epithelial cell line (Epi) were treated with alcohol (0 to 200 mM) or acetaldehyde (0 to 100 μM) in a cyclic fashion or incubated with supernatants collected from treated cells. Healthy cell %, reactive oxygen species (ROS), and proliferation were assessed via flow cytometry, luminescence, scratch wound, and colorimetric assays, respectively. A 15-plex multiplex assay was performed on cell supernatants, followed by IL-6 and IL-8 qRT-PCR and ELISA.
Results: Healthy HEMF decreased to less than 80% at 30 mM alcohol and 70 μM acetaldehyde, with microscopic changes at 40 μM acetaldehyde. HEMF ROS was detected at 100 mM alcohol and 80 μM acetaldehyde. Supernatants from 30 mM alcohol- or 40 μM acetaldehyde-treated HEMFs increased Epi proliferation more than two-fold that of lower doses. In the complementary studies, healthy Epi cells decreased to less than 80% at 50 mM and 70 μM acetaldehyde, with microscopic changes at 40 μM. Supernatants from Epi treated with 50 mM alcohol or 40 μM acetaldehyde increased HEMF proliferation more than two-fold that of lower doses. A multiplex assay of supernatants showed the greatest increase in concentrations of IL-6 and IL-8 in HEMFs and in Epi treated with higher doses of alcohol or acetaldehyde. Neutralization of IL-6 and IL-8 in supernatants of HEMFS and esophageal epithelial cells inhibited the proliferation of Epi and HEMFs, respectively.
Conclusions: Alcohol and acetaldehyde doses in which the majority of HEMFs and epithelial cells are healthy, elicit the production of paracrine mediators with pro-proliferative effects on neighboring cells. Understanding the effect of alcohol and acetaldehyde on HEMFs and HEMF-epithelial interactions could help to identify the molecular basis by which alcohol increases the risk for esophageal cancer.
Keywords: acetaldehyde; alcohol; esophageal epithelial cells; human esophageal myofibroblasts; paracrine; proliferation.
© 2023 The Authors. Alcohol: Clinical and Experimental Research published by Wiley Periodicals LLC on behalf of Research Society on Alcohol.