Ferroptosis inducer Erastin inhibits proliferation of liver cancer cells in vitro by down-regulating ACSL4

doi:10.12122/j.issn.1673-4254.2024.11.09

Abstract

Abstract:

Objective To investigate the expression of Acyl-CoA synthetase long-chain family member 4 (ACSL4) in liver cancer and its role in regulating ferroptosis and proliferation of liver cancer cells. Methods Clinical samples of liver cancer and adjacent normal liver tissues were examined for malondialdehyde (MDA) contents and for expressions of mRNA and protein expressions of ACSL4 and proliferating cell nuclear antigen (PCNA) using RT-qPCR and Western blotting. Human liver cancer Huh-7 cells were treated with Erastin (a ferroptosis inducer), Fer-1 (a ferroptosis inhibitor), or both, and the changes in expression levels of MDA, ACSL4 and PCNA were detected, and the cell proliferation was assessed with plate cloning assay. Results MDA contents were lower and ACSL4 and PCNA expressions were higher significantly in liver cancer tissues than in adjacent liver tissues. In Huh-7 cells, Erastin treatment significantly inhibited mRNA and protein expressions of ACSL4 and PCNA, suppressed cell proliferation, and increased MDA contents. Fer-1 alone did not produce significant effect on cell viability but reversed the effect of Erastin on ACSL4 and PCNA expressions, cell proliferation and MDA contents. Conclusion ACSL4 level is significantly overexpressed in liver cancer. Erastin increases MDA contents and down-regulates ACSL4 expression, thereby promoting ferroptosis and inhibiting proliferation of liver cancer cells, and these effects can be reversed by Fer-1.

Key words: liver cancer, cell proliferation, Erastin, Acyl-CoA synthetase long-chain family member 4, ferroptosis

Peipei ZHAO, Zhigang ZHOU, Yuanyuan YANG, Shusheng HUANG, Yixuan TU, Jian TU. Ferroptosis inducer Erastin inhibits proliferation of liver cancer cells in vitro by down-regulating ACSL4[J]. Journal of Southern Medical University, 2024, 44(11): 2131-2136.

Figures/Tables 5

Fig.1 HE staining and MDA contents detection in liver cancer and adjacent tissues. A: HE staining (Original magnification: ×40). B: MDA contents in the tissues (n=6). **P<0.01 vs Normal liver tissues.

Fig.3 Effects of Erastin and Fer-1 on viability of Huh-7 cells assessed using CCK-8 assay. A: Effects of different concentrations of Erastin on viability of Huh-7 cells (*P<0.05, **P<0.01, ****P <0.0001 vs 0 µmol/L). B: Effects of different concentrations of Fer-1 on viability of Huh-7 cells. C: Effects of Erastin combined with Fer-1 on viability of Huh-7 cells (**P<0.01, ****P<0.0001 vs Control; ##P<0.01 vs 0 µmol/L Fer-1).

Fig. 4 Effects of Erastin and Fer-1 on MDA contents and ACSL4 and PCNA expressions in Huh-7 cells. A: Effects of Erastin and Fer-1 on MDA contents in Huh-7 cells. B: Effects of Erastin and Fer-1 on mRNA expressions of ACSL4 and PCNA (RT-qPCR). C: Effects of Erastin and Fer-1 on protein expressions of ACSL4 and PCNA (Western blotting). D: Relative protein expression levels of ACSL4 and PCNA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Control; #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001 vs Erastin.

Fig.5 Effects of Erastin and Fer-1 on proliferation ability of Huh-7 cells. A: Plate cloning assay of the cells treated with Erastin and Fer-1. B: Number of cell clones. ***P<0.001 vs Control; ###P<0.001 vs Erastin.

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