Ethanol Modulation of Hypoxia Response Proteins in Oral Epithelial Cells

Methods:  Two oral cancer cell lines were utilised as a model of oral tissue for this study, Ca9.22 a gingival cell line and TR146 a buccal cell line. Cells were cultured in the presence or absence of a range of ethanol concentrations over 6, 24 and 48h time points. Cell viability was determined using Alamar Blue. Extracellular production of H₂O₂ was monitored using Amplex Red and intracellular levels were monitored using 2',7'-dichlorfluoresceindiacetate using flow cytometry and confocal microscopy. Propidium iodide staining prior to flow cytometry allowed for cell cycle analysis. Protein expression levels of targets of interest within the cell were determined through quantitative real time polymerase chain reaction (qPCR) and western blotting. 

Results:  Ethanol concentrations from 0.1-3% showed no detrimental effect on cell survival over the given time course. The presence of ethanol was found to reduce the release of H₂O₂ in a dose dependent manner (p< 0.5-0.001, n=3). Variation in expression levels of HIF1A occurred in tandem with the observed reduction in H₂O₂ levels. No significant induction of apoptosis was observed.

Conclusions: These findings suggest that treatment of carcinoma cell lines with ethanol causes a shift in the normal hypoxic state preventing apoptosis allowing low levels of H₂O₂ and other ROS species to enhance tumour cell survival.