Haiyingjie Lin1, Xiaoting Chen2, Chengyong Zhang2, Tingting Yang2, Zhendong Deng2, Yuwei Song3, Lanlan Huang2, Fuxiang Li2, Qingchu Li4, Shaoqiang Lin5, Dadi Jin6. 1. Department of Orthopedic, The Third Affiliated Hospital of Southern Medical University, No. 183 Zhongshan Dadao West, 510630, Guangzhou, China. 2. Clinical Department of Guangdong Metabolic Disease Research Centre of Integrated Chinese and Western Medicine, The First Affiliated Hospital of Guangdong Pharmaceutical University, No. 19 Nonglinxia Road, 510000, Guangzhou, China. 3. Central Laboratory of The First Affiliated Hospital of Jinan University, No. 613 Huangpu Dadao West, 510630, Guangzhou, China. 4. Department of Orthopedic, The Third Affiliated Hospital of Southern Medical University, No. 183 Zhongshan Dadao West, 510630, Guangzhou, China. Electronic address: lqc16@263.net. 5. Clinical Department of Guangdong Metabolic Disease Research Centre of Integrated Chinese and Western Medicine, The First Affiliated Hospital of Guangdong Pharmaceutical University, No. 19 Nonglinxia Road, 510000, Guangzhou, China. Electronic address: sqlin123@163.com. 6. Department of Orthopedic, The Third Affiliated Hospital of Southern Medical University, No. 183 Zhongshan Dadao West, 510630, Guangzhou, China. Electronic address: dadijin@yahoo.com.
Abstract
PURPOSE: EF24, a synthetic analogue of curcumin, was developed as an anti-tumor compound to induce apoptosis, inhibit proliferation and metastasis in various cancers. However, whether EF24 induces ferroptosis in osteosarcoma cells or not, and its underlying mechanism remains largely elusive. METHODS: After EF24 combining with or without other compounds treatments, mRNA expression profiles were proceeded by RNA sequencing. Cytotoxicity was measured by cell counting kit-8 assay. Cell death was quantified by flow cytometer. Gene expression was quantified by real-time PCR. Protein level was detected by western blot. Malonydialdehyde (MDA) level was measured by lipid peroxidation MDA assay kit. Reactive oxygen species (ROS) level was measured by ROS Assay Kit. Ferric ion was measured by Iron Assay kit. RESULTS: EF24 significantly induced cell death in osteosarcoma cell lines, and this effect was significantly reversed by ferrostatin-1, but not Z-VAD(Ome)-FMK, MRT68921 or necrosulfonamide. EF24 significantly increased MDA level, ROS level and intracellular ferric ion level, these effects were significantly attenuated by ferrostatin-1. EF24 upregulated HMOX1 expression in a dose dependent manner, overexpression of HMOX1 facilitated EF24 to induce ferroptosis in osteosarcoma cell lines. HMOX1 knockdown attenuated EF24-induced cytotoxicity and attenuated EF24-induced inhibition of Glutathione Peroxidase 4 (GPX4) expression. CONCLUSION: Our results showed that EF24 upregulated HMOX1 to suppress GPX4 expression to induce ferroptosis by increasing MDA level, ROS level and intracellular ferric ion level. Thus, EF24 might serve as a potential agent for the treatment of HMOX1-positive osteosarcoma patients.
PURPOSE:EF24, a synthetic analogue of curcumin, was developed as an anti-tumor compound to induce apoptosis, inhibit proliferation and metastasis in various cancers. However, whether EF24 induces ferroptosis in osteosarcoma cells or not, and its underlying mechanism remains largely elusive. METHODS: After EF24 combining with or without other compounds treatments, mRNA expression profiles were proceeded by RNA sequencing. Cytotoxicity was measured by cell counting kit-8 assay. Cell death was quantified by flow cytometer. Gene expression was quantified by real-time PCR. Protein level was detected by western blot. Malonydialdehyde (MDA) level was measured by lipid peroxidation MDA assay kit. Reactive oxygen species (ROS) level was measured by ROS Assay Kit. Ferric ion was measured by Iron Assay kit. RESULTS:EF24 significantly induced cell death in osteosarcoma cell lines, and this effect was significantly reversed by ferrostatin-1, but not Z-VAD(Ome)-FMK, MRT68921 or necrosulfonamide. EF24 significantly increased MDA level, ROS level and intracellular ferric ion level, these effects were significantly attenuated by ferrostatin-1. EF24 upregulated HMOX1 expression in a dose dependent manner, overexpression of HMOX1 facilitated EF24 to induce ferroptosis in osteosarcoma cell lines. HMOX1 knockdown attenuated EF24-induced cytotoxicity and attenuated EF24-induced inhibition of Glutathione Peroxidase 4 (GPX4) expression. CONCLUSION: Our results showed that EF24 upregulated HMOX1 to suppress GPX4 expression to induce ferroptosis by increasing MDA level, ROS level and intracellular ferric ion level. Thus, EF24 might serve as a potential agent for the treatment of HMOX1-positive osteosarcomapatients.