Herceptin induces ferroptosis and mitochondrial dysfunction in H9c2 cells

Sun,Lei; Wang,Hua; Yu,Shanshan; Zhang,Lin; Jiang,Jue; Zhou,Qi

doi:10.3892/ijmm.2021.5072

Herceptin induces ferroptosis and mitochondrial dysfunction in H9c2 cells

Authors:
- Lei Sun
- Hua Wang
- Shanshan Yu
- Lin Zhang
- Jue Jiang
- Qi Zhou
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Affiliations: Department of Ultrasound, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710004, P.R. China
Published online on: December 16, 2021 https://doi.org/10.3892/ijmm.2021.5072
Article Number: 17
Copyright: © Sun et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Ferroptosis has been previously implicated in the pathological progression of cardiomyopathy. Herceptin (trastuzumab), which targets HER2, is commonly applied for the treatment of HER2⁺ breast cancer. However, its clinical use is limited by its cardiotoxicity. Therefore, the present study aimed to investigate if targeting ferroptosis could protect against Herceptin‑induced heart failure in an in vitro model of H9c2 cells after treatment of Herceptin, Herceptin + ferroptosis inhibitor ferrostatin‑1 (Fer‑1) or Herceptin + Deferoxamine. H9c2 cell viability was measured by MTT assay. Reactive oxygen species (ROS) levels were detected by measuring the ﬂuorescence of DCFH‑DA‑A and MitoSOX™ Red. Glutathione (GSH)/oxidized glutathione (GSSG) ratio was measured using the GSH/GSSG Ratio Detection Assay kit. Mitochondrial membrane potential and ATP content were evaluated by JC‑1 staining and bioluminescent assay kits, respectively. Protein expressions of glutathione peroxidase 4, recombinant solute carrier family 7 member 11, mitochondrial optic atrophy1‑1/2, mitofusin, Acyl‑CoA synthetase long chain family member 4, cytochrome c, voltage‑dependent anion‑selective channel, dynamin‑related protein, mitochondrial fission 1 protein and mitochondrial ferritin were evaluated by western blotting. It was found that Herceptin reduced H9c2 cell viability whilst increasing intracellular and mitochondrial ROS levels in a dose‑ and time‑dependent manner. Furthermore, Herceptin decreased glutathione peroxidase (GPX) protein expression and the GSH/ GSSG ratio in H9c2 cells in a dose‑ and time‑dependent manner. The Fer‑1 abolished this Herceptin‑induced reduction in cell viability, GSH/GSSG ratio, mitochondrial membrane potential and ATP content. Fer‑1 also reversed the suppressive effects of Herceptin on the protein expression levels of GPX4, recombinant solute carrier family 7 member 11, mitochondrial optic atrophy1‑1/2 and mitofusin in H9c2 cells. Subsequently, Fer‑1 was found to reverse the Herceptin‑induced increase in mitochondrial ROS and iron levels in H9c2 cells, as well as the increased protein expression levels of Acyl‑CoA synthetase long chain family member 4, cytochrome c, voltage‑dependent anion‑selective channel, dynamin‑related protein, mitochondrial fission 1 protein and mitochondrial ferritin in H9c2 cells. However, compared with deferoxamine, an iron chelator, the effects of Fer‑1 were less effective. Collectively, these findings provided insights into the pathogenic mechanism that underlie Herceptin‑induced cardiomyopathy, which potentially provides a novel therapeutic target for the prevention of cardiotoxicity in HER2⁺ breast cancer treatment.

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