Shikonin inhibits triple-negative breast cancer-cell metastasis by reversing the epithelial-to-mesenchymal transition via glycogen synthase kinase 3β-regulated suppression of β-catenin signaling

Triple-negative breast cancer (TNBC) is characterized by elevated metastasis, low survival, and poor response to therapy. Although many specific and effective agents for treating TNBC have been investigated, promising therapeutic options remain elusive. Here, we screened the inhibitory activities of three main components of Lithospermum erythrorhizon Sieb. et Zucc (shikonin, acetylshikonin, and β,β-dimethylacrylshikonin) on TNBC cells. The results revealed that shikonin potently decreased the viabilities of TNBC MDA-MB-231 and 4T1 cells but showed less cytotoxicity to normal mammary epithelial MCF-12A cells. Additionally, shikonin reversed the epithelial-to-mesenchymal transition (EMT) in MDA-MB-231 and 4T1 cells. Shikonin depressed cell migration and invasion, upregulated E-cadherin levels, downregulated N-cadherin, vimentin, and Snail levels, and reorganized the cytoskeletal proteins F-actin and vimentin. Shikonin reversed EMT by inhibiting activation of β-catenin signaling through attenuating β-catenin expression, nuclear accumulation, binding to T-cell factor consensus oligos, and transcription of its targeted EMT-related genes. Moreover, shikonin upregulated glycogen synthase kinase 3β (GSK-3β) levels, leading to enhanced phosphorylation and decreased levels of β-catenin. Furthermore, shikonin administration significantly inhibited lung metastasis of MDA-MB-231 cells in NOD/SCID mice accompanied by low systemic toxicity. Histological analysis confirmed that shikonin elevated levels of E-cadherin, phosphorylated β-catenin, and GSK-3β, and decreased levels of vimentin and β-catenin in pulmonary metastatic foci. These results indicated that shikonin potently inhibits TNBC metastasis by targeting the EMT via GSK-3β-regulated suppression of β-catenin signaling, which highlights the importance of shikonin as a potential candidate for novel anticancer therapeutics against TNBC.