The Inhibitory Effects And Potential Regulatory Mechanisms Of Bruceine D On Proliferation,Migration,Invasion And Stem Cell-like Properties Of Osteosarcoma

Objective:Osteosarcoma is the most common primary malignant bone tumor with a relatively low incidence.Standardized chemotherapy combined with surgery have significantly improved the survival rate of osteosarcoma patients.However,those with osteosarcoma exhibiting resistance to chemotherapy or presenting with metastasis usually have a poor prognosis.Osteosarcoma stem cells(OSCs)are considered as a potential cause for tumor resistance,metastasis and recurrence,and may become a new promise for the treatment of osteosarcoma.Brucetin D(BD)is a natural active pharmaceutical ingredient derived from the fruit of Brucea javanica.It has a variety of biological functions,including anti-tumor effects.This study aims to explore the anti-osteosarcoma effects of BD,and investigate its underlying mechanisms.Methods:CCK-8 assays,CFSE staining and dilution assays,and colony formation assays were performed to detect cell viability and proliferation of osteosarcoma cells.PI staining and flow cytometry was used to detect the effect of BD on the cell cycle progression.Annexin V-PE / 7-AAD double staining and flow cytometry was used to investigate cell apoptosis.Wound-healing assay and Transwell chamber assay were applied to detect osteosarcoma cell migration and invasion ability.Western Blot were used to evaluate the expression of proteins which were involved in regulating cell cycle and apoptosis,STAT3 signal pathway,and stem cell properties.The effects of BD on the stemness of OSCs were assessed by sphere-forming assay and flow cytometry analysis of SP(Side Populations)cells and CD133~+ cells.Subcutaneous xenograft tumor model was used to evaluate the inhibitory effect and potential mechanisms of BD on osteosarcoma in vivo.Results:The results of CCK-8,CFSE staining,colony formation assays showed that BD significantly inhibited osteosarcoma cell viability and proliferation,and this inhibitory effect was time-and dose-dependent.BD could also induce cell cycle arrest.And the expressions of cyclin D1,CDK4,and CDK2 were significantly down-regulated.Furthermore,BD could induce apoptosis in osteosarcoma cells,and upregulated the expression of pro-apoptotic protein cleaved caspase 3,and correspondingly decrease the level of anti-apoptotic protein Bcl-2.Results of wound-healing and Transwell assays showed that BD significantly inhibited the migration and invasion ability of osteosarcoma cells;The expression of N-cadherin,MMP-2,and MMP-9 which play important role in tumor migration and metastasis.Upon BD treatment,the phosphorylation level of JAK2 / STAT3 was decreased,and the expression of SHP1 was upregulated.Moreover,STAT3 inhibitor Stattic inhibited the phosphorylation of STAT3,and the proliferation and migration ability of osteosarcoma cells by Western Blot,CCK-8,wound-healing,and Tranwell assays.Rescue assays showed that STAT3 pathway activator IL-6 could partially rescue the inhibitory effect of BD on cell proliferation and migration ability.In addition,results of sphere-forming assay,flow cytometry analysis of CD133 positive cells and SP cells,and expression of several stem cell markers showed that BD treatment inhibited the self-renewal ability of OSCs.In vivo,BD could significantly inhibit osteosarcoma growth without significant organ side effects.And,immunohistochemistry analysis showed that BD inhibited the STAT3 activity,as well as proliferation and metastasis potential of osteosarcoma.Conclusion:The results of this study indicated that BD exerts a strong inhibitory effect on tumor growth and stem cell-like properties of osteosarcoma which may be partially through STAT3 inhibition,suggesting that BD maybe a promising therapeutic candidate against osteosarcoma.