As a novel discovered myokine, irisin is considered to be a promising candidate for the treatment of metabolic disorders and cancer. However, little is known about the anti-metastasic effect of irisin on osteosarcoma cells and its underlying mechanisms. In the present study, we aimed to explore the effect of irisin on the migration and invasion of osteosarcoma cells and the underlying mechanisms involved. Viability and proliferation of osteosarcoma cells were examined by MTT assay. Then, by using scratch wound healing assay and Transwell assays, we evaluated migratory and invasive ability of the cells, respectively. Moreover, the expression of epithelial-to-mesenchymal transition (EMT) markers were determined by qPCR, western blot and immunofluorescence staining after treatment with IL-6 and irisin. Furthermore, the expression of ERK, p38, STAT3 and Snail were detected by western blot analysis. Finally, an inhibitor of STAT3, WP1066 was applied to testify the effect of irisin on the expression of EMT markers and Snail. It was found that irisin treatment significantly suppressed the proliferation, migration and invasion of osteosarcoma cells. Furthermore, irisin reversed the IL-6-induced epithelial-mesenchymal transition (EMT) in osteosarcoma cells by regulating the expression of E-cadherin, N-cadherin, vimentin, fibronectin, MMP-2, MMP-7 and MMP-9. In addition, irisin suppressed the IL-6-activated phosphorylation of STAT3 and the expression of Snail in osteosarcoma cells. Finally, blockade of STAT3 by WP1066 (a STAT3 inhibitor) further enhanced the effect of irisin on the EMT and Snail expression in osteosarcoma cells. Collectively, our findings revealed that irisin may play a critical role in the IL-6-induced EMT of osteosarcoma cells via the STAT3/Snail signaling pathway.

The current study investigates the interactive behavior of titanium alloy particle-challenged osteoblastic bone marrow stromal cells (BMSCs) and macrophage lineage cells in a murine knee-prosthesis failure model. BMSCs were isolated from male BALB/c mice femurs and induced in osteogenic medium. At 24 hours after isolation, BMSCs in complete induction medium were challenged with 1, 3, or 5mg/ml titanium particles for 7 days. Culture media were collected at 2, 4 and 6 days and cells were harvested at 7 days for alkaline phosphatase (ALP) assay/stains. Cell proliferation in the presence of Ti particles was periodically evaluated by MTT assay. Mice implanted with titanium-pin tibial implants were given an intra-articular injection of 50μl medium containing 5×105 Ti particles-challenged bone marrow derived osteoblastic cells, followed by a repeat injection at 2 weeks post-op. Control mice with titanium-pin implants received a naïve osteoblastic cell transfusion. After sacrifice at 4 week, the implanted knee joint of each group was collected for biomechanical pin-pullout testing, histological evaluation and RT-PCR analysis of mRNA extracted from the joint tissues. Ti-particles significantly stimulated the proliferation of BMSC-derived osteoblastic cells at both high and low particle concentrations (p<0.05), with no marked differences between the particle doses. ALP expression was diminished following Ti-particle interactions, especially in the high dose particle group (p<0.05). In addition, the culture media collected from short-term challenged (48 hours) osteoblasts significantly increased the numbers of TRAP+ cells when added to mouse peripheral blood monocytes cultures, in comparison with the monocytes cells receiving naïve osteoblasts media (p<0.05). Intra-articular introduction of the osteoblastic cells to the mouse pin-implant failure model resulted in reduced implant interfacial shear strength and thicker peri-implant soft-tissue formation, suggesting that titanium particles-challenged osteoblasts contributed to periprosthetic osteolysis. Comparison of the gene expression profiles among the peri-implant tissue samples following osteoblast injection did not find significant difference in RunX2 or Osterix/Sp7 between the groups. However, MMP-2, IL-1, TNF-α, RANKL, and TRAP gene expressions were elevated in the challenged-osteoblast group (p<0.05). In conclusion, titanium alloy particles were shown to interfere with the growth, maturation, and functions of the bone marrow osteoblast progenitor cells. Particle-challenged osteoblasts appear to express mediators that regulate osteoclastogenesis and peri-prosthetic osteolysis.