Terpinen-4-ol Inhibits Cutaneous Squamous Cell Carcinoma Cell Proliferation And Motility By Upregulating CAPN2 Expression

Objective:Terpinen-4-ol(T4O)is a major constituent of tea tree essential oil and other aromatic plants with anti-inflammatory,anti-aging and anti-tumor properties.Although T4 O has anti-proliferative and pro-apoptotic effects in melanoma and other tumor cells,its therapeutic effect on cutaneous squamous cell carcinoma(c SCC)is unclear.In this study,to observe the therapeutic effects of T4 O in c SCC.Methods:In this study,we used cck-8 assay and plate clone formation assay to examine the effect of T4 O on c SCC cell proliferation.Flow cytometry was used to detect the effect of T4 O on the cycle arrest situation and apoptosis of c SCC cells.the migration and invasion ability of c SCC cells after T4 O treatment was examined using scratch assay and Transwell assay.Meanwhile,EMT-related proteins were detected by immunofluorescence and Western blotting.In addition,the effect of T4 O on the proliferation ability of c SCC cells was examined in an in vivo nude mouse subcutaneous tumorigenesis model and immunohistochemical staining was performed.To further understand the targets of T4 O on c SCC,RNA sequencing and i TRAQ analysis were performed to find the differentially expressed m RNAs and proteins between T4O-treated and control DMSO-treated c SCC cells.And the detected proteins were further confirmed by Western blotting.Results:Through our study,we found that the action of T4 O on c SCC cells inhibited proliferation and clone formation and induced apoptosis in c SCC cells.Furthermore at the same time,T4 O treatment significantly inhibited the migration and invasion of c SCC cells and suppressed their epithelial-mesenchymal transition phenotype.Furthermore,similar to the results of in vitro assays,T4 O inhibited tumorigenesis of c SCC in vivo.To further understand the targets of T4 O action on skin squamous carcinoma,the investigators detected significant upregulation of CAPN2 expression at the m RNA and protein levels in T4O-treated c SCC cells compared with control DMSO treatment by RNA sequencing and i TRAQ analysis.It was further confirmed by Western blotting that T4 O significantly increased the expression of CAPN2 and also promoted the protein cleavage of two target proteins of CAPN2,β-catenin and caspase-12.Conclusions:These results suggest in summary that the antitumor activity of T4 O against c SCC is mediated by upregulation of CAPN2 expression and downregulation ofβ-catenin and caspase-12 expression.