| Objective:Osteosarcoma originates from mesenchymal tissue,mostly occurs in bone tissue.It is a common malignant bone tumor common in adolescents or children.There are more men than women.Its progression is fast,lung metastasis is early,prognosis is bad,relapse and even death.At present,the treatment of osteosarcoma includes comprehensive resection of the focus as far as possible through surgical procedures and the comprehensive adjuvant treatment of anti tumor during the perioperative period.As the first choice of chemotherapy,neoadjuvant chemotherapy can not only increase the safety of the operation,but also prepare the patient to customize the appropriate prosthesis.However,the limited variety of drugs currently used in chemotherapy is often accompanied by obvious systemic toxicity.It brings pain to patients,and it is imperative to find more effective and less side effects drugs and elucidate its action principle.Zoledronic acid is representative of the third generation,small side effects of bisphosphonates,which itself has a regulatory role in bone metabolism,inhibit osteoclast activity in vitro,further induce osteoclast apoptosis,and inhibit bone destruction caused by various reasons lead to increased bone resorption activity.Other studies have shown that zoledronic acid can also inhibit a variety of tumor cells or viscous matrix release stimulation factor induced osteoclast activity and bone calcium release,thus slowing the progression of bone metastasis and occurrence,and to a certain extent cause osteosarcoma cell death.PI3K/AKT signaling pathway is a signal transduction pathway that participates in the differentiation,growth and reproduction of most cells.AKT alias protein kinase B,after activation,continues to activate its downstream factor（GSK-3β,etc.）,which regulates cell cycle and apoptosis.Glycogen kinase 3βis the key glycogen synthetase,which relies on the phosphorylation of its substrates to regulate many important cell signaling pathways.A large number of studies have shown that the expression of GSK-3βin osteosarcoma is increased,and the apoptosis of osteosarcoma cells is changed after inhibiting the activity of GSK-3β.Wnt signaling pathway is involved in the differentiation and regeneration of bone marrow stromal cells,affecting the generation and differentiation of osteoblasts and osteoclasts,regulating bone repair,and playing a key role in bone growth,regeneration and remodeling.Wnt signaling pathway can induce the degradation complex ofβ-Catenin,including Axin,APC,CK1and GSK-3β.It makes the accumulation ofβ-Catenin and the freeβ-Catenin enter the nucleus and regulate gene expression.The aim of this study is to investigate the effect of zoledronic acid on the biological behavior of osteosarcoma cell line,and to investigate the effects of zoledronic acid on the important proteins and gene expression of PI3K-AKT and Wnt signaling pathways after osteosarcoma MG-63 and U-2 OS cell line.Methods:1.MTT method was used to detect MG-63 osteosarcoma cells viability when treated with 25,50,100,200μM ZOL for 24,48 and 72 h;50%inhibition rate of drug concentration time effect,during control group,experimental group,pure GSK-3βinhibitor group,GSK-3βinhibitors+experimental group cell viability control.2.scratch test clear osteosarcoma MG-63 cells apoptosis and invasion capacity when treated with25,50,100,200μM ZOL for 24,48 and 72 h;3.TEM observation of osteosarcoma MG-63 cells apoptosis change treated with zoledronic acid 50%in the inhibition rate of the drug concentration time（100 mol/L,48h）,during control group,experimental group,simple GSK-3βgroup,GSK-3βinhibitors+experimental group cell;4.flow cytometry of human osteosarcoma MG-63 and U-2 OS cell apoptosis change treated with zoledronic acid 50%in the inhibition rate of the drug concentration time（100 mol/L,48h）,during control group,experimental group,simple GSK-3βgroup,GSK-3βinhibitors+experimental group cell;5.Western blot method check osteosarcoma MG-63 and U-2 OS cells when treated with zoledronic acid 0,25,50,100,200 mol/L After the 48h,with the increase of drug concentration of phosphonic acid effect,the expression of P-AKT and P-GSK-3βprotein;detection of human osteosarcoma MG-63and U-2 OS cells when treated with zoledronic acid 100 mol/L After the 24,48,72h,with the increase of zoledronic acid treatment time,the expression of P-AKT and P-GSK-3βprotein situation detection;osteosarcoma MG-63 and U-2 OS cells treated with zoledronic acid 50%in the inhibition rate of the drug concentration time（100 mol/L,48h）,the expression ofβ-Catenin,c-Myc,Cyclin-D1 protein,during control group,experimental group,simple GSK-3βgroup,GSK-3βinhibitors+experimental group.Results:1.the results of MTT showed that osteosarcoma MG-63 cells increased with zoledronic acid treatment time and drug concentration,cell viability and apoptosis inhibition rate increased,50%inhibition rate of about 100 mol/L drug 48h;pure GSK-3βinhibitor group compared with the control group had no obvious difference,GSK-3βinhibitors+the experimental group results can inhibit cell viability,but compared with the same experimental group with the protection of the role of cell vitality.2.the results of scratch tests showed that after the treatment of zoledronic acid 0,25,50,100 and 200mol/L for 24,48 and 72h,the cell migration ability of osteosarcoma MG-63 cells was inhibited,and the cell apoptosis was changed.3.the results of TEM showed that the osteosarcoma MG-63 cells to effect of zoledronic acid 100 mol/L for 48h,control group and GSK-3βinhibitor group cell structural integrity;experimental group and experimental group+GSK-3βinhibitor cell apoptosis significantly,to effect the experimental group significantly.4.flow cytometry showed that osteosarcoma MG-63and U-2 OS cells to effect of zoledronic acid 100 mol/L for 48h,control group and GSK-3βinhibitor group cell mild apoptosis,experimental group and experimental group+GSK-3βinhibitor group cell apoptosis significantly,the apoptosis rate of experimental group higher drug.5.Western Blot showed the osteosarcoma MG-63 and U-2 OS cells to effect of zoledronic acid 100 mol/L for 48h,control group and GSK-3βinhibitor group cellβ-Catenin,c-Myc,Cyclin-D1 protein expression is equivalent;experimental group and experimental group+GSK-3βinhibitor group cellβ-Catenin,c-Myc and Cyclin-D1protein decreased and in the experimental group was significantly;osteosarcoma MG-63and U-2 OS cells in the zoledronic acid 0,25,50,100,200 mol/L 48h and 100 mol/L after 24,48,72h,the results show that with the increase of zoledronic acid treatment time and drug concentration,the expression of P-AKT and P-GSK-3βprotein reduce.Conclusion:1.zoledronic acid can inhibit osteosarcoma MG-63 cell growth and promote apoptosis in vitro;2.After zoledronic acid effect on osteosarcoma MG-63 and U-2 OS cells,GSK-3βdownstream proteinβ-Catenin,c-Myc,Cyclin-D1 expression decreased;the experimental group was given GSK-3βinhibitor Li2CO3,expression ofβ-Catenin,c-Myc,Cyclin-D1 than that of the experimental group increased,indicating zoledronic acid by GSK-3β/β-Catenin signaling pathway on apoptosis of osteosarcoma MG-63 and U-2 OS cells.3.zoledronic acid can decrease the expression of P-AKT and PGSK-3βand further down regulate the expression of downstream proteinβ-Catenin,c-Myc and Cyclin-D1,indicating that zoledronic acid can affect the apoptosis of osteosarcoma MG-63 and U-2 OS cells through AKT/GSK-3β/β-Catenin signaling pathway. |
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