Found programs: Anhui Provincial Academic and Technical Leaders and Reserve Candidates Project ( No . 2022H291) ; Open Project of Key Laboratory of Anti-inflammatory and Immune Medicine , Ministry of Education , at Anhui Medical University (No . KFJJ-2021-04) ; Commercial Research Funds of Anhui Medical University (No . 2023009) ; Research Project of Anhui Provincial Institute of Translational Medicine (No . 2023zhyx-C24)
Authors：Zhu Xinyue1 , Ren Qiaohui 1 , Zang Yan1 , Zhou Xinyi 1 , Yao Junxiao1 , Wang Lianzi 1 , Sha Xudong2 , Li Tao1
Keywords：hepatocellular carcinoma; Erastin; RSL3 ; ferroptosis; in vitro model
DOI：10.19405/j.cnki.issn1000-1492.2025.04.004
〔Abstract〕 Abstract Objective To establish an in vitro model of ferroptosis induced by Erastin and RAS-selective lethal 3 (RSL3) in hepatoma cells , and to provide theoretical basis for the development of novel therapeutic strategies for HCC . Methods Hepatoma cells ( HCCLM3 , HepG2 , Hep3B , Huh7 and PLC/PRF/5 ) in logarithmic growth phase were treated with Erastin (0 - 40 μmol/L) and RSL3 (0 - 10 μmol/L) at double concentrations respective- ly . After 24 h , CCK-8 method was used to detect cell viability , draw growth curve , calculate IC50 , and HCC cells sensitive to inducers were selected for follow-up experiments . The effect of inducer on the state of hepatoma cells was observed under light microscope , and immunoblotting and flow cytometry were used to verify whether the fer- roptotic model in vitro was successfully constructed . Results Huh7 , Hep3B and HepG2 cells were sensitive to Erastin and RSL3 , but HCCLM3 and PLC/PRF/5 were insensitive to Erastin and RSL3 . When the concentration of Erastin and RSL3 reached the maximum , the survival rate was still above 65% . Huh7 , Hep3B and HepG2 cells were selected for subsequent experiments . Compared with the control group , the expression of Glutathione peroxi- dase 4 (GPX4) , a ferroptotic marker , was down-regulated in a concentration-dependent manner. In Huh7 , Hep3B and HepG2 cells , lipid reactive oxygen species ( ROS) levels significantly increased after 24 h treatment with 10 μmol/L and 20 μmol/L Erastin , respectively; in Huh7 cells , lipid ROS levels significantly increased after 24 h treatment with 0. 5 μmol/L and 1 μmol/L RSL3 , respectively; in Hep3B and HepG2 cells , lipid ROS levels sig- nificantly increased after 24 h treatment with 1 μmol/L and 2 μmol/L RSL3 , respectively , compared with control group . Conclusion Huh7 , Hep3B and HepG2 cells are highly sensitive to Erastin and RSL3 . Huh7 , Hep3B and HepG2 cells treated with 10 μmol/L Erastin for 24 h are good models for simulating ferroptosis induced by Erastin in vitro , Huh7 cells treated with 0. 5 μmol/L RSL3 for 24 h and Hep3B and HepG2 cells treated with 1 μmol/L RSL3 for 24 h are good models for simulating ferroptosis induced by RSL3 in vitro .