The Anti-tumor Function of Shikonin by Targeting EGFR/AKT/mTOR Signaling in Human Osteosarcoma Cells

Background: Shikonin, a purified naphthoquinone separated from a Traditional Chinese medicinal herb Lithospermum erythrorhixon, which exhibits anticancer properties.Objective: To clarify the molecular mechanisms of therapeutic effects of shikonin against osteosarcoma. Methods: Cell Counting Kit-8 (CCK-8) assay was employed to evaluate cell viability. Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) double-staining was conducted to evaluate the apoptotic ratio of the MG-63 cells. The effects of shikonin on the invasiveness of osteosarcoma cells were implemented by a transwell cell migration assay. In the meantime, a western blot assay was employed to detect alterations in the relevant mechanism proteins within osteosarcoma cells. Molecular docking analysis was conducted to anticipate the binding interaction between shikonin and EGFR/protein kinase B (AKT)/mTOR. Results: We observed that shikonin suppressed proliferation and induced apoptosis in the MG-63 cells in a dose-dependent manner. Pursuing these findings, the potential molecular mechanisms were detected. Shikonin intervention blocked epidermal growth factor receptor (EGFR) phosphorylation and decreased epidermal growth factor (EGF)-induced activation of downstream signaling molecules, such as AKT and mammalian target of rapamycin (mTOR) in the MG-63 cells. However, the additional recombinant human epidermal growth factor (rHuEGF) could stimulate the activation of EGFR/AKT/mTOR signaling and reverse cell apoptosis caused by shikonin. Molecular docking analysis showed that shikonin presented the highest bonding ability with EGFR, AKT, and mTOR. Conclusion: Our results show that shikonin inhibits human osteosarcoma development via inactivating EGFR/AKT/mTOR signaling. It demonstrates that shikonin may act as a potential therapeutic agent in osteosarcoma treatment.