Study On The Effects And Mechanisms Of PTN In The Drug Resistance Of Osteosarcoma

BACKGROUNDOsteosarcoma(OS)is the most common primary bone malignancy in children and adolescents.As a quickly proliferative and highly invasive sarcoma,OS has often metastasized by the time it is diagnosed.Although the treatment of OS has achieved encouraging progress in the past decades,chemoresistance is still a major hindrance to successful treatment of OS.Pleiotrophin(PTN)is a neurotrophic growth factor that is related to diverse biological properties including cell differentiation,proliferation,migration and angiogenesis.Studies have revealed that PTN is involved in the tumorigenesis and tumor progression,and is differentially expressed in good and poor responders to chemotherapy in OS patients.Studies also reported that PTN is associated with the chemoresistance of neuroblastoma.However,there are limited available data about the role of PTN in OS and whether PTN might be involved in affecting the chemoresistance of OS remains elusive.OBJECTIVETo explore the effects of PTN on drug resistance of OS cells and the underlying mechanisms.METHODS(1)qRT-PCR and Western-blot were employed to detect the expression of PTN in drug resistant and parental cell lines.(2)Immunohistochemistry was used to explore the PTN expression in OS tissues and analyze the relation between PTN expression and clinical parameters.(3)Drug sensitivity assay,colony formation assay and xenograft assay were used respectively to detect the effect of PTN on the chemoresistance of OS and further explore the underlying molecular mechanisms.RESULTS(1)PTN was highly expressed in drug-resistant MG63/DOX cells compared with the parental MG63 cells.(2)In vitro study revealed that PTN knockdown decreased the cellular chemoresistance to doxorubicin in MG63,MG63/DOX and U2 OS cells and this effect could be reversed by incubating with rh PTN.(3)In DOX-treated OS cells,silencing PTN upregulated the apoptosis rate and inhibited clone formation ability,while PTN overexpression showed the opposite effects.(4)In a xenograft model,silencing and overexpressing PTN respectively increased and reduced the cellular sensitivity to doxorubicin.(5)Further molecular assays revealed PTN upregulated the expression of anaplastic lymphoma kinase(ALK),pGlycogen Synthase Kinase(GSK)3β,β-catenin and P-glycoprotein(P-gp).The rescue assays with the β-catenin inhibitor and MDR1/P-gp inhibitor showed PTN promoted drug resistance to DOX in OS cells by activating ALK/GSK3β/β-catenin signaling pathway,thereby upregulating P-gp.(6)Clinical respective study indicated that PTN expression was associated with necrosis rate and local recurrence.High PTN expression correlated with poor overall and disease-free survival(DFS),and was identified as an independent adverse prognostic factor for DFS.CONCLUSIONPTN enhanced the drug resistance to doxorubicin in OS by upregulating P-gp through activating ALK/GSK3β/β-catenin signaling pathway.PTN could be a promising prognostic biomarker for OS patients and downregulating PTN may be used as an effective strategy to overcome drug resistance in OS treatment.