Xanthoxyletin Inhibits Proliferation of Human Oral Squamous Carcinoma Cells and Induces Apoptosis, Autophagy, and Cell Cycle Arrest by Modulation of the MEK/ERK Signaling Pathway.

BACKGROUND This study aimed to investigate the effects of xanthoxyletin, a plant-derived coumarin, on human oral squamous cancer cells in vitro and in mouse xenografts in vivo. MATERIAL AND METHODS The study included SCC-1 human oral cancer cells and EBTr normal embryonic bovine tracheal epithelial cells, which were treated with 0 µM, 5 µM, 10 µM, and 20 µM of xanthoxyletin for 24 hours. The MTT assay assessed cell viability, and autophagy was detected by electron microscopy. Cell apoptosis was investigated using 4',6-diamidino-2-phenylindole (DAPI), annexin V, and propidium iodide (PI) fluorescence flow cytometry, which was also used to investigate the cell cycle. Protein expression was measured by Western blot. Mouse xenografts were used for the in vivo evaluation of the effects of xanthoxyletin. RESULTS Xanthoxyletin significantly inhibited the proliferation of oral cancer cells (IC₅₀, 10-30 µM) with lower cytotoxicity for normal cells. Xanthoxyletin treatment was associated with G2/M arrest of the cell cycle and with increased apoptosis and autophagy of SCC-1 cells. Apoptosis and autophagy induced by xanthoxyletin were also associated with changes in expression of the apoptosis-associated proteins, Bax and Bcl-2, and the autophagy-associated proteins, LC3I, LC3II, Beclin 1, p62, and VSp34. Xanthoxyletin inhibited the expression of components of the signaling cascade of the MEK/ERK pathway in the SCC-1 oral cancer cells. The in vivo effects of xanthoxyletin showed inhibition of growth of mouse xenografts. CONCLUSIONS Xanthoxyletin inhibited the proliferation of human oral squamous carcinoma cells and induced apoptosis, autophagy, and cell cycle arrest by modulation of the MEK/ERK signaling pathway.