Shikonin Inhibits Oral Cancer Progression Through Suppression of Metastasis and Angiogenesis and Induction of Autophagy and Apoptosis

Background: Shikonin, a compound extracted from Lithospermum erythrorhizon, has demonstrated therapeutic effects on cancer; however, its effects on oral cancer remain unclear. Objectives: This study aimed to explore the therapeutic value of shikonin for the treatment of oral cancer. Methods: MTT, colony formation, and Transwell assays were employed to evaluate the inhibitory effects of shikonin on the proliferation and migration abilities of human oral cancer cell lines (SCC-4 and SAS). Apoptosis and cell viability were assessed using the TdT-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay. To investigate the potential anticancer mechanisms of shikonin, RNA sequencing, quantitative PCR, and western blotting were performed to analyze changes in the expression levels of oral cancer cell-related genes and proteins (matrix metalloproteinases-2 (MMP-2), matrix metalloproteinases-9 (MMP-9), VEGF-A, VEGF-C, Beclin-1, autophagy-related genes (ATG5), and light chain-3 (LC-3)). Additionally, animal xenograft experiments were conducted to examine the in vivo antitumor effects of shikonin. Results: The findings revealed that the external application of shikonin specifically targeted oral cancer cells without affecting normal cells and led to a dose-dependent inhibition of their growth. Even at non-lethal doses, shikonin effectively suppressed the production of metalloproteinases, thereby inhibiting cancer cell migration and wound healing. Furthermore, shikonin treatment reduced levels of tumor progression factors, such as vascular endothelial growth factor (VEGF)-A and VEGF-C, which are released during the early stages of cancer cell angiogenesis and lymphangiogenesis. Meanwhile, higher doses of shikonin induced cell autophagy and activated proteins such as ATG-5, LC-3B, and Beclin-1. At lethal doses, shikonin further decreased mitochondrial membrane potential, released calcium ions, and triggered apoptotic pathways. However, the administration of a calcium ion chelator (BAPTA-AM) inhibited shikonin-induced apoptosis. Conclusions: These results demonstrate that shikonin induces autophagy and activates apoptotic pathways in oral cancer cells. Shikonin treatment significantly inhibited oral cancer growth and induced apoptosis in a rat model. In conclusion, shikonin effectively inhibited oral cancer cell growth, metastasis, and the expression of tumor progression-related proteins. Given the ease of drug delivery to the affected area in oral cancer, shikonin holds substantial potential for future applications that may improve patient recovery and enhance cure rates.