Thymol Modulation of Androgen Receptor in Oral Squamous Cell Carcinoma

Objectives: Oral Squamous Cell Carcinoma (OSCC) occurs two to three-fold more in males over females. Reports of androgen receptor (AR) expression are conflicting; some indicate overexpression in up to 67% in OSCC and others report decreased expression. While some studies indicate that AR inhibition decreases OSCC oncogeneic behavior, studies of head and neck SCC reveal improved metastases-free and overall survival rates with AR expression. Thymol has anti-proliferative effects on OSCC in vitro, anti-tumor effects in vivo, anti-androgenic properties, and is shown to decrease AR mRNA expression in odontoblasts. The goal of this study is to determine if thymol regulates AR expression/activation in OSCC and if this regulation is androgen dependent or independent.
Methods: OSCC cell lines Cal27 and HSC3 were treated with thymol, dihydrotestosterone (DHT), antiandrogens bicalutamide and MDV, and gefitinib (EGFR inhibitor). Quantitative PCR and western blot analysis of AR expression/activation in vitro and immunohistochemistry (IHC) of AR expression in thymol-treated Cal27-derived tumors were performed. Cell proliferation and migration assays were completed to determine if AR operates in an androgen dependent or independent manner in OSCC.
Results: AR mRNA expression was confirmed in Cal27 and HSC3 cell lines. Thymol significantly reduced AR mRNA expression in both cell lines and induced a dose-dependent decrease in cell viability. Notably, no change in cell proliferation/migration was seen in Cal27 or HSC3 cells upon treatment with DHT, bicalutamide and/or MDV. Western blot analysis revealed that AR protein is not expressed in OSCC cell lines (Cal27, HSC3, SCC4, SCC9, SCC25). Furthermore, no definitive AR protein expression was noted via IHC.
Conclusions: While thymol downregulates AR mRNA in OSCC and reduces cell proliferation and tumor growth, AR protein is not expressed in all OSCC lines tested. Therefore, AR signaling does not play a role in OSCC and thymol’s anti-tumor effects are via an AR-independent mechanism.