Chronic Ethanol Exposure Induces Early Epithelial-to-Mesenchymal Transition (EMT) and Premalignant Changes in Gingival Keratinocytes: An In Vitro Model of Very Early Oral Carcinogenesis

Early molecular events underlying ethanol-induced oral squamous cell carcinoma development remain insufficiently understood, primarily due to a lack of suitable in vitro systems that recapitulate the initial stages of premalignant transformation. Therefore, a cell culture model of human gingival keratinocytes representing progressive stages of early ethanol-induced cell transformation was established and comprehensively characterized. The three cell lines, named "gingival keratinocytes" (GK), "epithelioid" (EPI) and "fibroblastoid" (FIB), and their derivatives were analyzed by morphological, cell biological and biochemical methods, with an emphasis on epithelial-to-mesenchymal transition (EMT)-related signaling pathways. All cell lines were non-tumorigenic in vitro. Chronic ethanol exposure induced distinct morphological and molecular alterations that capture early premalignant changes in vitro. This includes reduced <i>E-Cadherin</i> and enhanced <i>Vimentin</i> expression, accompanied by an increased production of reactive oxygen species. Notably, even morphologically stable cell lines displayed metabolic susceptibility to EMT induction, indicating the early activation of transformation-associated signaling cascades even in a premalignant state. These alterations, however, closely mirrored pathohistological features of oral squamous cell carcinomas such as loss of epithelial integrity and acquisition of mesenchymal characteristics. Collectively, the presented model provides a robust and accessible in vitro platform for investigating very early ethanol-induced oral carcinogenesis mechanisms that are relevant in a premalignant state and may facilitate the identification of diagnostic and preventive biomarkers to improve patient outcomes in alcohol-associated oral cancer and precursor lesions.