Terpinen-4-ol suppresses proliferation and motility of cutaneous squamous cell carcinoma cells by enhancing calpain-2 expression

Terpinen-4-ol (T4O), a key constituent of tea tree essential oil and various aromatic plants, has shown promising antiproliferative and pro-apoptotic effects in melanoma and other cancer types. However, its efficacy against cutaneous squamous cell carcinoma (cSCC) remains unclear. Thus, in this study, we investigated the in vivo and in vitro effects of T4O on cSCC cell lines and preliminarily explored its impacting pathways.

Using CCK8 and assay colony formation, we assessed the viability of cSCC A431, SCL-1, and COLO-16 cells treated with T40 at varying concentrations (0, 1, 2, and 4 μM). Flow cytometry was employed to evaluate T4O’s effect on cSCC cell’s cycle progression and apoptosis induction. Additionally, western blotting was utilized to examine the expression intensities of N-cadherin and E-cadherin, two indicative markers of the epithelial-mesenchymal transition (EMT) pathway. T4O’s in vivo effect on inhibiting tumor progression was evaluated on an established xenograft tumor model. Then, the molecular mechanisms of T4O’s antitumor effect were explored by an integrated genome-wide transcriptomics and proteomics study on cSCC A431c cells. Finally, calpain-2’s potential mediator role in T4O’s anti-tumor mechanism was investigated in calpain-2 knockdown cell lines prepared via siRNA transfection.

It’s demonstrated that T4O treatment inhibited cSCC proliferation, clonogenicity, migration, and invasion while inducing apoptosis and suppressing the EMT pathway. T4O administration also inhibited cSCC tumorigenesis in the xenograft tumor model. RNA-sequencing and iTRAQ analysis detected significant upregulation of calpain-2 expression in T4O-treated cSCC cells. Western blotting confirmed that T4O significantly increased calpain-2 expression and promoted proteolytic cleavage of β-catenin and caspase-12, two calpain-2 target proteins. Importantly, siRNA-mediated calpain-2 knockdown relieved T4O’s suppressive effect on cSCC cell proliferation and motility. Mechanistically, T4O upregulates calpain-2 expression and promotes the cleavage of β-catenin and caspase-12, with siRNA-mediated calpain-2 knockdown mitigating T4O’s suppressive effects.

These findings suggest that T4O’s antitumor activity in cSCC is mediated through the upregulation of calpain-2 expression and subsequent modulation of β-catenin and caspase-12.