Thymol inhibits oral squamous cell carcinoma growth via mitochondria‐mediated apoptosis

Background Thymol is a transient receptor potential ankyrin subtype 1 channel, ( TRPA 1) agonist found in thyme and oregano. Thymol has antioxidant, anti‐inflammatory, and antimicrobial properties; thus, thymol is added to many commercially available products including Listerine mouthwash. Thymol is also cytotoxic to HL ‐60 (acute promyelocytic leukemia) cells in vitro. Therefore, we evaluated the effects of thymol against oral squamous cell carcinoma ( OSCC ) and its anticancer mechanism‐of‐action. Methods The antiproliferative effects of thymol in OSCC Cal27 cells were determined by MTS assays. Antitumor effects were evaluated in Cal27‐ and HeLa‐derived mouse xenografts. Calcium imaging, mitochondrial transmembrane potential (ΔΨm) studies, and Western blot analysis of cleaved PARP (c‐ PARP ) evaluated thymol's mechanism‐of‐action. Results Thymol had significant, long‐lasting antiproliferative effects in vitro. In vivo, thymol displayed significant antitumor effects in Cal27‐derived tumors. Thymol's anticancer effects were confirmed in HeLa‐derived xenografts demonstrating that thymol effects are not tumor‐type specific. Calcium imaging verified calcium influx in Cal27 cells that were reversed with the TRPA 1 antagonist, HC 030031. However, no calcium influx was seen in HeLa cells indicating that TRP channels do not regulate thymol cytotoxicity. This was confirmed using cell viability assays in which pre‐treatment with HC 030031 had no effect on thymol cytotoxicity. Instead, ΔΨm studies revealed that thymol induces significant ΔΨm depolarization and apoptosis. Conclusion Our findings provide the first evidence of thymol's novel antitumor effects against OSCC in vivo, which do not rely on TRPA 1 activity. Instead, we show that thymol induces mitochondrial dysfunction and apoptosis and may be efficacious against multiple cancers.