Secretome Analysis of Oral Keratinocytes Chronically Exposed to Shisha

Cancer Biomark. 2019;25(1):29-41. doi: 10.3233/CBM-182099.


Background: Shisha smoking has been associated with multiple diseases including oral cancer. However, a mechanistic study to investigate alteration of secreted proteins in oral cells due to shisha smoking is lacking.

Objectives: Elucidation of differentially secreted proteins by immortalized human normal oral keratinocytes (OKF6/TERT1) upon chronic exposure to shisha.

Methods: OKF6/TERT1 was chronically treated with 0.5% shisha extract for 8 months. Conditioned media from shisha treated (OKF6/TERT1-Shisha) and untreated (OKF6/TERT1-Parental) cells were subjected to TMT-based quantitative proteomic analysis. Bioinformatics analysis of differentially secreted proteins was carried out using SignalP, SecretomeP and TMHMM. Immunoblot validation of selected proteins was carried out to confirm the proteomics results.

Results: Proteomic analysis of OKF6/TERT1-Parental and OKF6/TERT1-Shisha secretome resulted in the identification of 1,598 proteins, of which 218 proteins were found to be differentially secreted (⩾ 1.5-fold; p-value ⩽ 0.05) in shisha treated cells. Bioinformatics analysis using prediction tools showed secretory potential of differentially secreted proteins identified in OKF6/TERT1-Shisha. Western blotting validated the expression of AKR1C2, HSPH1 and MMP9 in OKF6/TERT1-Shisha secretome in agreement with proteomic data.

Conclusion: This study serves as a useful resource to understand the effect of chronic shisha smoking on the milieu of secreted proteins of oral cells. In vivo studies are warranted to supplement our in vitro data to elucidate the role of these proteins as early diagnostic biomarkers for oral carcinogenesis among shisha smokers.

Keywords: OKF6/TERT1; TMT; oral cancer; proteomics; secretory biomarkers; waterpipe.

MeSH terms

  • Biomarkers, Tumor / metabolism
  • Cell Line
  • Computational Biology
  • Humans
  • Keratinocytes / drug effects*
  • Keratinocytes / metabolism*
  • Mouth Neoplasms / chemically induced
  • Mouth Neoplasms / metabolism
  • Plant Extracts / toxicity
  • Proteome / drug effects*
  • Proteome / metabolism
  • Proteomics
  • Tobacco, Waterpipe / toxicity*


  • Biomarkers, Tumor
  • Plant Extracts
  • Proteome