Effects of Sanguinarine on Gene Expression and Carcinogen Metabolism in Oral Keratinocytes

Epidemiologic studies have shown an association between use of sanguinarine containing products and development of precancerous oral lesions. Although sanguinarine structurally resembles polycyclic aromatic hydrocarbons (PAHs-known carcinogens) no studies have established that oral mucosa can metabolize and bioactivate sanguinarine. Purpose: This study investigated the effects of sanguinarine on cultured human oral keratinocytes. Previous studies from our laboratory have confirmed that these cell lines retain expression and function of carcinogen metabolizing enzymes. Methods: These studies assessed sanguinarine's effects on: 1) cell proliferation (MTT mitochondrial reduction assay), 2) carcinogen metabolism [effects on bioactivation of the tobacco associated PAH, benzo-a-pyrene (BaP), HPLC analyses], 3) cellular activation of the aryl hydrocarbon receptor (AhR) via EMSAs, 4) cellular expression of the carcinogen metabolizing AhR responsive genes [cytochrome P450 1A1 and 1B1, NADPH quinone reductase, glutathione-S-transferase alpha, glutathione peroxidase] by RT-PCR. Results: Cell dose response studies show: 1) IC50s of approximately 0.75 mM in all cell lines (n=5), 2) 0.5 mM sanguinarine resulted in comparable cell numbers and viabilities as control cultures (n=5). Isolated CYP 1A1 and 1B1 human eyzyme metabolism studies (0.1, 1, and 10 mM sanguinarine) showed a dose-dependent inhibition of BaP bioactivation. Relative to the control enzyme preparations, all sanguinarine doses inhibited 1A1 BaP bioactivation, while only the 10 mM dose inhibited 1B1 BaP bioactivation (p<0.05, Neuman-Keuls Multiple Comparison test, n=3 for each group). Initial EMSA results show that sanguinarine causes activation, nuclear translocation and DNA binding of the AhR. Preliminary RT-PCR studies show that sanguinarine upregulates expression of the potential bioactivating CYP 1A1 and 1B1 enzymes. Conclusions: Our data suggest that sanguinarine functions: i) as a competitive substrate for CYP 1A1 and 1B1 enzymes, and therefore is potentially capable of being bioactivated by these enzymes, ii) as a ligand for the AhR and increases expression of AhR responsive enzymes. PO1 DE 12704.