The Mechanism Of Curcumin Analogue EF24 Induces Ferroptosis In Osteosarcoma Cells Through HMOX-1

BACKGROUND:Osteosarcoma(OS)is the most common primary malignant bone tumor in adolescents and children.Chemotherapy plays a key role in the treatment of osteosarcoma.The 5-year survival rate of osteosarcoma patients undergoing surgery combined with chemotherapy is 61%,while the 5-year survival rate of osteosarcoma patients receiving surgery alone is only 11%.The key to OS chemotherapy lies in the patients' response to chemotherapy.The 5-year survival rate of patients with tumor necrosis rate exceeding 90%after neoadjuvant chemotherapy is reach up to 90%;the 5-year survival rate of patients with tumor necrosis below 90%is only 50%-60%.However,because chemotherapeutic drugs are biologically toxic,simply increasing the intensity of chemotherapy does not help extend the survival of patients.Therefore,the development of new low-toxic and highly effective chemotherapy drugs is an important research direction in the field of OS treatment.As a representative curcumin analogue,EF24 has good biosafety,broad-spectrum antitumor activity,and fixes the defect of curcumin's poor bioavailability.However,it is unclear whether EF24 produces antitumor activity by inducing ferroptosis and whether EF24 induces cytotoxicity in OS cells.Ferroptosis is a type of regulated cell death(RCD)catalyzed by iron and characterized by membrane lipid peroxidation.Ferroptosis inducers can induce Ferroptosis by inhibiting the protective mechanism of cell peroxidation,or increasing iron ions in cells.Because tumor cells need to rapidly proliferate,they have higher requirements for iron than normal cells.This makes tumor cells more sensitive to iron death than normal cells when iron homeostasis is destroyed.Therefore,Ferroptosis inducers have potential advantages in antitumor treatments.In this study,we used EF24 to treat osteosarcoma cells,detect the cytotoxicity and the changes in gene expression profiles,systematically characterize ferroptosis-related phenotypes,and construct cell-line models in order to reveal the type of EF24-mediated osteosarcoma cell death as well as the mechanism and provide a scientific theoretical basis for the forthcoming clinical use of EF24PURPOSE:To investigate the type and mechanism of EF24-mediated osteosarcoma cell death.METHODS:(1)Detecting the cytotoxicity induced by EF24 in OS cells by CCK-8 and flow analysis with fluorescent staining,then compare it with curcumin;(2)Screening gene expression profiles of EF24-mediated OS cells by transcriptome sequencing technology;(3)Treating OS cells wit EF24 and RCD inhibitors,then iron ion,reactive oxygen species(ROS)and lipid oxidation levels were detected for characterizing the types of cell death mediated by EF24(4)The effect of EF24 on the expression level of HMOX-1 gene was detected by qRT-PCR and WB;(5)HMOX-1 overexpressing OS cell model was constructed by lentivirus transfection,and its effect on EF24-induced ferroptosis and cell proliferation ability were detected by CCK-8;(6)The expression of HMOX-1 in OS samples and its paired normal tissues was detected by qRT-PCR and WB.RESULTS:(1)EF24 significantly induces cytotoxicity in OS cells,and it is higher when comparing with the same concentration of curcumin;(2)Analysis of differentially expressed genes after gene sequencing showed that EF24-induced cytotoxicity was associated with ferroptosis;(3)EF24-induced OS cell death can be reversed by an ferroptosis inhibitor,but cannot be reversed by apoptosis,cell death,and autophagy inhibitors;(4)EF24 up-regulates intracellular iron level,ROS level,and lipid oxidation level of OS cells,and this effect can be reversed by an ferroptosis inhibitor;(5)EF24 up-regulated HMOX-1 gene expression in OS cells in a dose-dependent manner;(6)Overexpression of HMOX-1 increases the sensitivity of OS cells to EF24-induced ferroptosis;(7)HMOX-1 expression is relatively elevated in OS samples.CONCLUSION:Our results shows that EF24 can act as an ferroptosis inducer to mediate ferroptosis and elevates the expression of HMOX-1 in a dose-dependent manner in OS cells By constructing HMOX-1-overexpression OS cell models,we demonstrated that high expression level of HMOX-1 promotes EF24-induced ferroptosis.Moreover,the relatively high expression of HMOX-1 in OS tissues of OS patients may make them more sensitive to the treatment of EF24.Thus we reveals the potential of EF24,a curcumin analogue with low toxicity to normal cells and broad spectrum anti-tumor activity,to be used in the treatment of OS in the near future.