Establishment of an in vitro model for Erastin and RSL3-induced ferroptosis in hepatocellular carcinoma
10.19405/j.cnki.issn1000-1492.2025.04.004
- Author:
Xinyue Zhu
1
;
Qiaohui Ren
1
;
Yan Zang
1
;
Xinyi Zhou
1
;
Junxiao Yao
1
;
Lianzi Wang
1
;
Xudong Sha
2
;
Tao Li
1
Author Information
1. Dept of Clinical Laboratory，The First Affiliated Hospital of Anhui Medical University，Hefei 230022
2. Dept of Pharmacology and Chemical Biology，Shanghai Jiao Tong University School of Medicine，Shanghai 200025
- Publication Type:Journal Article
- Keywords:
hepatocellular carcinoma;
Erastin;
RSL3;
ferroptosis;
in vitro model
- From:
Acta Universitatis Medicinalis Anhui
2025;60(4):611-618
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish anin vitromodel 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 respectively. 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 ferroptotic modelin vitrowas 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 peroxidase 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 significantly 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 Erastinin 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 RSL3in vitro.
- Full text:2026012609293321159Erastin和RSL3诱导肝癌铁死亡体外模型的构建_朱欣悦.pdf
