Effects Of Dihydrotanshinone ?(DHI) On Human Osteosarcoma And Its Mechanism

Objective: To investigate the inhibitory effect of dihydrotanshinone I(DHI)in human osteoscarcoma and its mechanism.Methods: The effect of different concentrations of DHI on proliferation in osteosarcoma cells(MG-63,143 B,Saos2,U2OS)was detected by Crystal violet assay,and the effect of DHI on the proliferation of human osteosarcoma cells(MG-63,143B)was further verified by MTT assay.The effects of different concentrations of DHI on clone formation,migration,invasion and apoptosis in osteosarcoma cells(143B,MG-63)were detected by Cloning formation assay,Transwell assay,Wound healing assay,Hoechst 33258 staining and flow cytometry.After treatment with different concentrations of DHI(Control,0,2,4,6?M),the protein expression levels of PCNA,Snail,matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-7(MMP-7),matrix metalloproteinase-9(MMP-9),N-Cadherin,Bcl-2,PARP,Cleaved–PARP,Caspase-3,Cleaved Caspase-3,LRP6,?-Catenin,Cyclin D1,c-Myc were detected by Western blot assay and the activity of Wnt/ ?-Catenin signaling pathway in osteosarcoma143 B cells was detected by Luciferase reporter assay.In order to furtherverify the effect of DHI on osteosarcoma in vitro,we established an orthotopic OS tumour animal model,The orthotopic OS tumour animal model was treated with DHI(5 mg / kg,15 mg / kg,25 mg / kg)Once every two days,the size of osteosarcoma was measured every 2 days.After21 days,the nude mice were killed and the expression levels of PCNA,N-Cadherin,Bcl-2,?-Catenin in tumor were detected by immunohistochemistry.Results: Crystal violet assay suggested that DHI can decreased the proliferation of osteoscarcoma cells(MG-63?143B?Saos2?U2OS)(P<0.05).MTT assay,Cloning formation assay,Transwell assay,Wound healing assay,Hoechst 33258 staining and flow cytometry showed that DHI can decreased proliferation,clone formation,migration,invasion of osteoscarcoma cells(MG-63?143B)(P<0.05)and induced apoptosis(P<0.05).In DHI group,Luciferase reporter assay suggested that the activity of Wnt/?-Catenin signaling pathway decreased significantly(P<0.05),and Western blot assay suggested that up-regulated the expressions of PARP,Cleaved–PARP,Cleaved Caspase-3 and Caspase-3,and down-regulated the expressions of PCNA,Snail,matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-7(MMP-7),matrix metalloproteinase-9(MMP-9),NCadherin,Bcl-2,LRP6,?-Catenin,Cyclin D1,c-Myc(P < 0.05).The results of in vitro experiment showed that the growth rate of tumor was slowed down in DHI treated group,and the expression levels of PCNA,Bcl-2,N-Cadherin and ?-Cadherin(P<0.05)were inhibited in DHI-treated group by immunohistochemistry.Conclusion: DHI inhibits the proliferation,migration and invasion of osteosarcoma 143 B cells and induces apoptosis,possibly by blocking Wnt/?-Catenin signaling pathway.