SDF-1/CXCR4 Promotes F5M2 Osteosarcoma Cell Migration By Activating The Wnt/?-catenin Signaling Pathway

BackgroundOsteosarcoma is the most common primary malignant bone tumor, mainly occurring in children and adolescents. The use of neoadjuvant chemotherapy in combination with orthopedic surgery has increased the long-term survival rates that now approach 70% in patients who have no metastatic OS at diagnosis. However, the outcome is more awful with a survival rate of <30% at 5 years in patients with metastatic or recurrent disease at present. Therefore, to understand how the signaling invoved in cell proliferation, invasion and migration of osteosarcoma is a key way for developing more effective tumor therapies.SDF-1 is a member of the CXC chemokine subfamily, and it interacts specifically with the only known ligand for the seven-transmembrane G protein-coupled receptor CXCR4. It has been shown that the interaction between SDF-1 and CXCR4 activates a lot of signaling pathway to play physiological functions, such as embryonic development, angiogenesis, hematopoiesis, heart development. Many studies have shown that there is abnormally high expression of CXCR4 in a variety of solid tumors and hematologic malignancies. Recent studies have shown that SDF-1/CXCR4 axis is related to growth, invasion and metastasis of tumor.Wnts are very conservative cysteine-rich protein on biological evolution. Wnt signaling pathway plays a role in regulating development and maintaining adult homeostasis by regulating cell proliferation, cell differentiation, cell migration, and apoptosis. Abnormal activation of the Wnt signaling pathway is involved in tumor invasion and migration. In recent years, more and more evidence proved that SDF-1/CXCR4 axis may affect tumor invasion and migration through the Wnt signaling pathway. However, concerning the relationship between SDF-1/CXCR4 axis and Wnt signaling pathway is not clear in osteosarcoma. Thus, this experiment studied the role of SDF-1/CXCR4 axis in osteosarcoma cell F5M2 migration and the relationship with the Wnt signaling pathway. ObjectiveTo investigate whether the functional SDF-1/CXCR4 signaling mediates chemotaxis in F5M2 OS cells as well as the underlying the relationship with the Wnt signaling pathway. Methods1.Using immunohistochemical technique to detect the expression of CXCR4 and ?-catenin in 60 cases of osteosarcoma and 10 cases of osteochondroma, and analyzing their relationship and clinical significance in osteosarcoma.2.The expression of CXCR4 and ?-catenin were determined in osteosarcoma cell F5M2 and normal osteogenesis cell h FOB1.19 by Western-blot with quantitative Real Time PCR.3. Transwell assays were performed to determine SDF-1-induced F5M2 migration.4. Immunofluorescence microscopy, Transwell assays and Western-blot were performed to reveal the relationship between SDF-1/CXCR4 axis with Wnt signaling pathway in osteosarcoma cell F5M2. Results1.The expression of CXCR4 protein was detected in 86.7%(52/60) cases of osteosarcoma and 20%(2/10) cases of osteochondroma, while ?-catenin expression was detected in 60%(36/60) case of osteosarcoma and in 10%(1/10) case of osteochondroma. The expression of CXCR4 was associated with clinical Ennecking stage and the metastasis of osteosarcoma(P <0.05), while the expression of ?-catenin was associated with clinical Ennecking stage and the metastasis of osteosarcoma(P <0.05). In addition, CXCR4 expression was positively correlated with ?-catenin experssion(P <0.05).2. Western-blot results showed that CXCR4 and ?-catenin expression level were higher in F5M2 cells than their expression in h FOB1.19 cells. QRT-PCR illustrated that CXCR4 expression is 6.5 times in F5M2 cells compared to h FOB1.19 cells, and ?-catenin expression is 8.1 times in F5M2 cells compared to h FOB1.19 cells.3.Transwell assays results showed that SDF-1 promoted F5M2 cell migration, which was abrogated by preincubation with AMD3100.4.Confocal immunofluorescent microscopy revealed that level of expression of ?-catenin was significantly higher after incubation with SDF-1, and the expression of ?-catenin did not alter with the incubation with AMD3100. Transwell assays results showed that SDF-1 promoted F5M2 cell migration, which was abrogated by preincubation with AMD3100 and FH535. Western-blot results showed that phosphorylation of AKT and ?-catenin increased in F5M2 cells pretreated with SDF-1. However, phosphorylation assays were performed in F5M2 cells pretreated with AMD3100 and LY294002, which showed that AMD3100 induced a decrease in SDF-1-induced phosphorylation of AKT and ?-catenin. Conclusion1.CXCR4 and ?-catenin presented a positive higher expression in osteosarcoma. The expression of CXCR4 and ?-catenin were related with clinical Ennecking stage and the metastasis of osteosarcoma. In addition, CXCR4 expression was positively correlated with ?-catenin expression.2.CXCR4 and ?-catenin expression level were higher in F5M2 cells than their expression in h FOB1.19 cells.3.SDF-1 promoted F5M2 cell migration by binding to CXCR4.4.SDF-1 promoted F5M2 cell migration by activating the AKT and Wnt/?-catenin signaling pathway, which was abrogated by preincubation with AMD3100 and LY294002.