Effects Of Botulinum Toxin Type A On Proliferation,migration And Apoptosis Of Human Osteosarcoma MG63 Cells Line And Its Mechanism

Backgrounds Osteosarcoma（OS）is an osteoblastic stem cell carcinoma and is one of the most common causes of cancer-related death in children.BoNT/A（botulinum toxin type A）is one of the most widely used drugs in clinical practice,mainly for the treatment of dystonic diseases.With the deepening of research on BoNT/A,some studies have found that BoNT/A has antitumor effects.BoNT/A inhibits tumor cells by regulating tumor cell apoptosis,proliferation and migration.Whether BoNT/A has an antitumor effect on osteosarcomas and which specific molecular and signaling pathways are involved in this process still need to be investigated.Objectives To investigate whether BoNT/A can regulate apoptosis,proliferation and migration of osteosarcoma MG63 cells and to preliminarily explore the molecular mechanism and signalling pathways involved..Methods 1.The human osteosarcoma MG63 line cells were treated with BoNT/A（0.25,0.5,1.0,2.0,4.0 U/ml）for 24 h,48h and 72 h,and proliferative capacity was detected by CCK8 assay.Calculate the IC50（half inhibitory concentration）at different time points and select the optimal concentration and duration of action of BoNT/A based on this.The degree of inhibition of the migratory ability of human osteosarcoma MG63 cells by BoNT/A can be detected by the scratch healing assay.Whether BoNT/A can induce apoptosis of osteosarcoma MG63 cells and whether the rate of apoptosis is related to the concentration of BoNT/A can be detected by FCM（flow cytometry）.2.IF（immunofluorescence）was used to detect the expression of synaptic vesicle protein SV2,which is responsible for the specific recognition and transport of BoNT/A into the cytoplasm of human osteosarcoma MG63 cell line.3.Western blot was used to detect the expression of apoptosis-related proteins,proliferation proteins,migration proteins,EMT（epithelial-mesenchymal transformation）related proteins and key proteins of the Wnt/β-catenin pathway in human osteosarcoma MG63 cells treated with BoNT/A.Results 1.CCK8 assay showed that the proliferation of human osteosarcoma MG63 cells was inhibited by BoNT/A and was positively correlated with time and concentration of BoNT/A.The effect of BoNT/A on the migration of human osteosarcoma MG63 cells was observed in the scratch healing experiment.The FCM results showed that BoNT/A promoted the apoptosis of the human osteosarcoma MG63 cell line,and the apoptosis rate of the human osteosarcoma MG63 cell line was positively correlated with the BoNT/A concentration.2.IF results showed that the synaptic vesicle protein SV2 was expressed on the cell membrane of human osteosarcoma MG63 cell line.3.After human osteosarcoma MG63 cells were treated with different concentrations of BoNT/A,the expression of proliferation-related protein PCNA was down-regulated by Western blot.The expression of migration-related proteins MMP2 and MMP9 was downregulated,the expression of EMT-related protein N-cadherin was inhibited and the expression of E-cadherin was increased.The expression of the apoptosis-related proteins Caspase3,Caspase9 and the pro-apoptotic protein Bax was promoted,while the expression of the anti-apoptotic protein Bcl-2 was inhibited.β-catenin,c-Myc and Cyclin D1 were key protein molecules of the Wnt/β-catenin pathway,and their expression was decreased.Conclusions 1.Human osteosarcoma MG63 cells express synaptic vesicle protein SV2 on the cell membrane.2.BoNT/A downregulated the expression of PCNA and inhibited the proliferation of osteosarcoma MG63 cells.3.BoNT/A inhibited the migration of human osteosarcoma MG63 cells by inhibiting the migration-related proteins MMP2 and MMP9 and blocking the EMT process.4.BoNT/A upregulated the expression of apoptosis-related proteins Caspase3,Caspase9 and Bax and downregulated the expression of Bcl-2,inducing apoptosis of osteosarcoma MG63 cells.5.BoNT/A may inhibit the Wnt/β-catenin signaling pathway and block the EMT process to induce apoptosis of osteosarcoma MG63 cells and inhibit cell proliferation and migration,thereby exerting an anti-tumor effect.