Gamabufotalin suppressed osteosarcoma stem cells through the TGF-β/periostin/PI3K/AKT pathway

To investigate the effect of gamabufotalin (GBT) on metastasis and modulation of stemness features in osteosarcoma, and the molecular mechanisms underlying such effects.

Human osteosarcoma U2OS/MG-63 cell lines were used in this study. Cell proliferation, migration, and invasion were determined by MTT assay, wound healing assay, and cell invasion assay, respectively. The inhibitive effect of GBT on stemness was assessed by flow cytometry and mammosphere formation. The protein levels of related proteins were detected by western blotting analysis. The effect of GBT on tumorigenicity and metastasis was determined by immunofluorescence staining and immunohistochemistry in vivo experiments.

We found that GBT suppressed the viability of U2OS/MG-63 cells in a time- and dose-dependent manner. Notably, GBT had no effect on the viability of human fetal osteoblastic (hFOB) 1.19 cells. Moreover, GBT increased the width of wounds, reduced the number of invasive osteosarcoma cells and reversed the epithelial-mesenchymal transition phenotype. Notably, we found that, compared with hFOB1.19 cells, the levels of transforming growth factor-β (TGF-β), periostin, phosphorylated-AKT (p-AKT), and phosphorylated-PI3K (p-PI3K) were higher in spheroids group than in parent cells. In addition, GBT reduced the ratio of CD133+ cells, the size of spheroids and Nanog, as well as the protein levels of SRY-box transcription factor 2 (SOX2), and octamer-binding protein 3/4 (OCT3/4). Our in vivo experiments showed that GBT consistently reduced lung metastasis lesions, the expression levels of matrix metalloproteinase 2 (MMP2), TGF-β, periostin, p-AKT, and p-PI3K (immunohistochemistry staining), as well as that of CD133 in tumor tissues (immunofluorescence analysis). From a mechanistic point of view, exogenous TGF-β/periostin/PI3K/AKT overexpression neutralized the reduction of GBT-decreased invasion/migration and the suppression of stemness properties.

Collectively, our data demonstrated that GBT inhibited the viability and tumorigenesis capability of osteosarcoma cells by blocking the TGF-β/periostin/PI3K/AKT signaling pathway. Therefore, GBT may represent a promising therapeutic agent for the management of osteosarcoma.