华蟾素注射液通过TFR1促进U2OS骨肉瘤细胞铁死亡的作用机制研究

Cinobufacini injection targeting TFR1 promotes osteosarcoma cells ferroptosis

目的通过体外实验揭示CI通过促进细胞铁死亡发挥治疗骨肉瘤的作用机制。方法使用的骨肉瘤细胞为U2OS细胞株,使用柠檬酸铁铵建立细胞外铁超载环境,CCK-8试剂盒检测细胞活性,EdU细胞增殖实验和划痕实验检测细胞增殖效率和迁移情况。使用ROS荧光探针对细胞内ROS含量进行定量分析,使用钙黄绿素染色检测细胞内铁离子含量情况,使用q-RTPCR和Western Blotting检测铁死亡相关靶标的基因及蛋白表达情况。结果在铁超载的环境下,CI能降低骨肉瘤细胞活性,并且显著降低肿瘤细胞的增殖效率;细胞迁移速率随着华蟾素注射液的浓度增加而减少。细胞内的铁离子、ROS和lipid-ROS的表达水平与CI的浓度存在正相关性。q-RTPCR及WB结果显示,CI可以抑制U2OS细胞中Glutathione Peroxidase 4(Gpx4)的表达水平并促进transferrin receptor 1(TFR1)的表达水平。结论华蟾素注射液能促进骨肉瘤细胞的铁死亡,Gpx4和TFR1可能是其作用靶点。

Objective Osteosarcoma(OS)is a common cause of cancer-related death in children and adolescents.Ferroptosis,the regulated cell death(RCD)other than apoptosis,has a close link and adolescents.Ferroptosis,the regulated cell death(RCD)other than apoptosis,has a close link with OS.Cinobufacini injection(CI)is a usual anti-tumor drug from Traditional Chinese Medicine(TCM)especially in treating OS.However,no researchers have studied whether CI induces ferroptosis in OS cells.We aimed to indicate that CI suppresses OS cells due to inducing ferroptosis.Methods CCK-8 assay was used to determine cell viability.EdU cell proliferation assay and wound healing assay were adopted to indicate the ability of cell proliferation and migration.Cytosolic ROS were stained for quantification.Cellular labile iron staining was evaluated with calcein-acetoxymethyl ester(calcein-AM).RT-qPCRand western blot were performed to evaluate the expression level of GPX4,HO-1,TFR1,FPN,ACSL4.Results After iron was accumulated,CI decreased the activity of U2OS cells and significantly reduced the proliferation efficiency of tumor cells.The cell migration rate decreased with the increase of the concentration of bufosin injection.The expression level of iron ion,ROS,and lipid-ROS in cells is positively correlated with the concentration of CI.The results of q-RTPCR and WB showed that CI could inhibit the expression of Glutathione Peroxidase 4(Gpx4)and promote the expression of transferrin receptor 1(TFR1)in U2OS cells.Conclusion CI can promote ferroptosis of osteosarcoma cells,and Gpx4 and TFR1 may be its targets.