Nicotine-Driven Modulation of MTOR Pathway in Oral Squamous Cell Carcinoma: a Cell-Specific Response to E-Cig Vapor Exposure

Objectives: Oral squamous cell carcinoma (OSCC) is a common disease affecting 30,000 people in the United States each year. The rise of electronic cigarettes (eCigs) has introduced a new, largely unregulated product line within the smoking industry. Although eCigs are generally considered to be less harmful than traditional smoking, little is known about their actual health effects. eCigs work by heating oil-based liquids and delivering aerosolized flavors, with or without nicotine, yet the health impacts of this mechanism remain under-researched. mTOR is a protein that regulates cell growth in response to nutrients and growth factors. The mTOR pathway includes other proteins such as p70 and 4EBP1, which influence cell invasion by increasing protein synthesis via distinct pathways. Our objective was to determine the role of mTOR pathway proteins in OSCC cells in culture.
Methods: Gingiva-derived Ca9-22 cells and tongue-derived Cal-27 cells were exposed to eCig vapor extract (EVE) generated by bubbling Green Apple flavored eCig solution, with or without nicotine, using a mod atomizer. The cells were exposed for 6 hours, after which protein lysates and conditioned media were immediately isolated. Immunoblot analysis was performed to examine mTOR pathway proteins.
Results: Immunoblotting of Ca9-22 cells showed: 1) An 11-fold increase in phosphorylated mTOR (p-mTOR) with nicotine;2) A 2-fold increase in phosphorylated p70 (p-p70), which decreased in the presence of nicotine; 3) A 3-fold decrease in phosphorylated 4EBP1 (p-4EBP1), independent of nicotine presence; and 4) A 3-fold decrease in phosphorylated AKT (p-AKT) when nicotine was present. Immunoblotting of Cal-27 cells showed: 1) A 16-fold increase in p-mTOR with nicotine; 3) A 4-fold decrease in p-4EBP1 with nicotine and
4) A 4-fold increase in p-AKT with nicotine.
Conclusions: We conclude that the activation of mTOR in Ca9-22 OSCC cells is correlated with increased mTOR protein and a decrease in p70 protein in a nicotine-dependent manner. Cal-27 cells showed increased mTOR protein and decreased 4EBP1 protein, also dependent on nicotine presence. Together, these findings suggest a cell-specific and nicotine-dependent response in the mTOR pathway in OSCC cells.