Identification of Shisha-Induced Molecular Alterations in Oral Keratinocytes

Objectives: Oral squamous cell carcinoma is a global health problem with a 5-year survival rate of OSCC patients is <60%. It is associated with many risk factors including smoking tobacco via the use of cigarettes, bidis and shisha. Shisha smoking is widely believed to be a hazard-free habit. Studies have reported shisha smoking to be associated with carcinomas of the lung, esophagus, bladder, and pancreas. In addition, Centers for Disease Control and Prevention, US lists shisha smoking to be associated with oral cancer. However, the mechanism of action of shisha on the oral keratinocytes has not been studied. A better understanding of this underlying mechanism may contribute to the identification of biomarkers for targeted therapy and better prognosis of the disease. In this study, we have established a chronic model of shisha-exposed oral keratinocytes to study the effect of shisha smoking on oral cells.

Methods: Non-neoplastic oral keratinocytes, OKF/TERT1, were chronically treated with shisha extract for 8 months. In vitro cellular assays as well as total proteomic analysis were performed using the OKF6/TERT1 parental (OKF6/Parental) and shisha extract-treated cells (OKF6/Shisha) to understand the effect of shisha smoking on cellular transformation.

Results: Chronic exposure of OKF6/TERT1 cells with shisha resulted in a significant increase in cellular proliferation, cell invasion and colony forming ability compared to the OKF6/Parental cells. Quantitative proteomic analysis of OKF6/Parental and OKF6/Shisha cells resulted in the identification of more than 4,000 proteins. Of these, more than 200 proteins were found to be dysregulated (1.5-fold) in the OKF6/Shisha cells compared to the OKF6/Parental cells.

Conclusions: This study reveals that chronic treatment of OKF6/TERT1 cells with shisha leads to cellular transformation. Elucidation of dysregulated proteins in shisha smoke-exposed cells will provide insights into the effect of shisha smoking on oral cells and aid in identification of therapeutic targets.