Tirapazamine Suppresses Osteosarcoma Cells Through SLC7A11 Mediated Ferroptosis

Part I Tirapazamine inhibits the proliferation and migration of osteosarcoma cells under hypoxia conditionsObjective To verify the effect of tirapazamine on osteosarcoma cells under normoxia and hypoxia.Methods Three-gas incubator was used to simulate hypoxia in vivo,and the effects of tirapazamine were explored through MTT,Transwell assay,and wound-healing experiments.Results Tirapazamine reduced the cell viability of 143B,MNNG/HOS and U2OS cell lines in a dose-dependent manner under hypoxia at 24 h.Tirapazamine could significantly reduce the ability of clone formation of 143B osteosarcoma cells,and as the concentration of tirapazamine increased,the number of clones-decreased more significantly.The number of cells that migrated to the lower chamber gradually decreased under hypoxia,but the cells under normoxia were not significantly affected.The migration ability of 143B cells treated with 10μM tirapazamine was significantly reduced under hypoxia,but the cells under normoxia were not significantly inhibited.Conclusion Tirapazamine could inhibit the proliferation and migration of osteosarcoma cells under hypoxia conditions.Part II Tirapazamine inhibits the proliferation and migration of osteosarcoma cells through ferroptosisObjective To explore the mechanism of Tirapazamine in inhibiting osteosarcoma cells.Methods In this part,the expression of reactive oxygen species,Fe²⁺accumulation,MDA and ferroptosis protein were detected by flow cytometry,fluorescence microscope,multi-functional microplate reader,and the like.Results As the concentration of tirapazamine increased,the amount of reactive oxygen species gradually increased.However,this phenomenon seemed not to be obvious under normoxia.The content of MDA under hypoxia increased along with the tirapazamine concentration,which was also significantly higher than that of MDA under normoxia.Tirapazamine at 10μM can significantly increase the accumulation of Fe²⁺in cells under hypoxia,whereas this phenomenon cannot occur under normoxia.Western blot experiment showed that the expression of p53 protein that inhibits tumors increased,while the antioxidant SLC7A11 and GPX4 proteins decreased significantly under hypoxia conditions.Conclusion Tirapazamine induce ferroptosis of osteosarcoma cells under hypoxia Part III Tirapazamine inhibits the proliferation and migration of osteosarcoma by inducing Ferroptosis through SLC7A11Objective To verify whether Tirapazamine induces ferroptosis and inhibits osteosarcoma cells through SLC7A11.Methods In this part,SLC7A11 was overexpressed first,and osteosarcoma cells were co-treated with tirapazamine.The effect of SLC7A11 was verified by cell clone formation experiment,Transwell assay,and reactive oxygen species.Results In this study,overexpressed SLC7A11 143B cells were treated with tirapazamine at a concentration of 10μM under hypoxia for 24 h.Cell clone formation experiments were used to verify the effect of tirapazamine on the proliferation of 143B cells.Compared with the individual tirapazamine group,SLC7A11 overexpressed combined tirapazamine group increased the number of clone formations,which was yet less than that of the SLC7A11 overexpressed group.The number of overexpressed SLC7A11 cells migrating to the lower chamber under hypoxia significantly increased,and more cells migrated to the lower chamber in the co-treatment group with tirapazamine than that in the individual tirapazamine group.Although the content of reactive oxygen species was significantly increased after Tirapazamine treatment,it could also be significantly restored after overexpression of SLC7A11.Conclusion Tirapazamine induce ferroptosis of osteosarcoma cells through inhibiting the expression of SLC7A11 under hypoxia conditions.