Metformin is an oral drug that has been widely used to treat type 2 diabetes mellitus. Interestingly, accumulated evidence indicate that metformin may reduce the risk of cancer in patients with type 2 diabetes and inhibit tumor cell growth and survival in numerous malignancies, including osteosarcoma (OS) cells. In the present study, we aimed to investigate the effects of metformin on the proliferation, migration, invasion, and sphere formation in OS MG63 cells in vitro. Metformin suppressed OS MG63 cell proliferation in a dose- and time-dependent manner and markedly blocked anti-metastatic potentials, migration, and invasion, by downregulating matrix metalloproteinase 2 (MMP2) and MMP9. Besides, we established OS cancer stem-like cell (CSC) model with sarcosphere formation assay and demonstrated that metformin posed damage on CSCs in OS by inhibiting sphere formation and by inducing their stemness loss. The stemness of CSCs in OS such as self-renewal and differentiation potentials was both impaired with a significant decrease of Oct-4 and Nanog activation. Consistent with this, the positive rates of CD90, CD133, and stage-specific embryonic antigen-4 (SSEA-4) were all observed with reductions in response to metformin exposure. In addition, Western blot showed that metformin activated AMPKα at Tyr172, followed by a downregulated phosphorylation of mammalian target of rapamycin (mTOR)/S6 and feedback activation of p-AKT Ser(473) in both OS MG63 cells and CSCs. This indicates that AMPK/mTOR/S6 signaling pathway might be involved in the growth inhibition of both OS MG63 cells and CSCs. These results suggest that metformin, a potential anti-neoplastic agent, might make it a novel therapeutic choice for the treatment of OS in the future.
Keywords: AMPK/mTOR/S6 signaling pathway; Cancer stem-like cells; Metastasis; Metformin; Osteosarcoma.