The Inhibitory Effect Of Lycorine On Osteosarcoma Cells And Underlying Mechanism

Objective: To analyze the inhibitory effect of lycorine on human osteosarcoma(OS)cells and its potential molecular mechanism.Methods: CCK8 assay was used to test the semi-inhibitory concentration(IC50)of lycorine on human OS cells(143B,MG63,SaoS2,U2OS)and human normal cells(MIHA,HK-2,HEB,HS-5).The effect of lycorine on the proliferation of OS cells was determined by crystal violet staining and clonoy formation assay.Cell cycle arrest was detected by flow cytometry.The ability of cell migration and invasion were performed by the scratch healing assay and transwell assay.Hoechst 33258 staining and flow cytometry were used to detect apoptosis.Western blot was used to analyze the cell proliferation related protein PCNA,cell cycle protein CylinD1 and CylinE1.Transformation of epithelial mesenchymal(EMT)related indexes Snail,Vimentin,N-cadherin,E-cadherin;Extracellular matrix(ECM)degradation related indexes MMP-2,MMP-7 and MMP-9.Mitochondrial apoptosis pathway involved proteins Bcl-2,Bax,Bad,Fadd,Caspase8 and Caspase9;death receptor apoptosis related proteins Caspase3,cleaved-Caspase3,PARP,and cleaved-PARP.Then,luciferase reporter gene system was applied to confirm the transcriptional activity of multiple signaling pathways,and western blot was used to detect the expression of β-Catenin and the downstream target molecule c-Myc in the Wnt/β-catenin signaling pathway,as well as the total ERK1/2,p-ERK1/2(T202/Y204),total AKT and p-AKT(S473)in the ERK1/2/MAPK and PI3K/AKT signaling pathways.Finally,orthotopic tumor transplantation model of OS in nude mice was constructed and tumor growth was observed.Pulmonary metastasis of OS was detected by H&E staining.The expression of PCNA,N-cadherin,Bcl-2,β-Catenin and p-ERK1/2(T202/Y204)and p-AKT(S473)were detected by IHC analysis.Results: Crystal violet staining,CCK8,flow cytometry,and clonoy formation assay revealed that lycorine significantly inhibited the proliferation of OS cells 143 B,MG63,SaoS2 and U2 OS,but had less toxicity to normal human cells MIHA,HK-2,HEB and HS-5.Lycorine could suppress the migration and invasion by scratch healing and Transwell assay,and induce apoptosis by Hoechst 33258 staining and flow cytometry assay.Luciferase reporter gene assay indicated that the transcriptional activity of Wnt/β-catenin,ERK1/2/MAPK and PI3K/AKT signaling pathways was decreased after lycorine treatment.Western blot demonstated lycorine reduced the expression of proliferation related protein PCNA,blocked the cell cycle at the G1/S phase and down-regulated CylinD1 and CylinE1;The expression levels of migration and invasion related indicators including Snail,Vimentin,N-cadherin,MMP-2,MMP-7 and MMP-9 were decreased,while E-cadherin was increased.The level of anti-apoptotic protein Bcl2 was decreased,and the apoptotic protein indicators Bax,Bad,Fadd,Caspase8,Caspase9,Caspase3,cleaved-Caspase3,PARP,and cleaved-PARP were increased.The protein level of total ERK1/2 and total AKT were not significantly changed,but the levels of p-ERK1/2(T202/Y204)and p-AKT(S473)were strikingly decreased.In orthotopic tumor transplantation model,lycorine distinctly inhibited tumor growth and distant pulmonary metastasis of OS.IHC results showed that lycorine could reduced the level of PCNA,N-cadherin,Bcl-2,β-Catenin,p-ERK1/2(T202/Y204)and p-AKT(S473).Conclusion: Lycorine may inhibit the proliferation,migration and invasion of human osteosarcoma cells and promote apoptosis by blocking the Wnt/β-catenin,ERK1/2/MAPK and PI3K/AKT signaling pathways.