ROS and iron homeostasis dependent ferroptosis play a vital role in 5-Fluorouracil induced cardiotoxicity in vitro and in vivo

Abstract

5-Fluorouracil (5-FU) was a key chemotherapeutic agent in the treatment of different solid tumors. However, cardiotoxicity was included among the therapeutic strategies of 5-FU. The molecular mechanism of cardiotoxicity induced by 5-FU remains unclear. The aim of the study was to investigate whether ferroptosis was involved in 5-FU-induced cardiotoxicity in vivo and in vitro. The in vivo cardiotoxicity model was induced by intraperitoneal injection of 5-FU at the dose of 15, 30, 60 mg/kg for 7 days. Body weight, general condition and plasma enzyme activities of the mice were observed to evaluate heart function. In addition, HE staining, MASSON staining and TEM technology was used. Western-blot analysis were performed to evaluate the protein level of iron transport, iron storage and reactive oxygen species (ROS) of ferroptosis. In H9c2 cardiomyocyte cells, cell viability, generation of ROS, mitochondrial activity and cellular Fe²⁺ levels were measured. The in vivo results showed that 5-FU significantly impaired cardiac function and structure. The serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels were significantly increased in 5-FU group. HE and MASSON staining showed that 5-FU caused structural injuries. In addition, 5-FU increased the level of ferroptosis markers involving malonaldehyde (MDA) and Fe²⁺ content. Ferrostatin-1 (Fer-1) was an aromatic amine that specifically binds with lipid ROS and protects cells against lipid peroxidation. Furthermore, 5-FU markedly induced ferroptosis in H9c2 cardiomyocyte cells, which mainly embodied as declined cell vitality, accumulated iron, elevated lipid peroxides. Conversely, inhibition of ferroptosis by Fer-1 completely abolished 5-FU-induced effects. Both in vivo and in vitro experiments indicated that 5-FU increased the expression of ferroptosis, mainly by reducing the expression of glutathione peroxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1), but enhancing the expression of transferrin receptor 1 (TfR1). In conclusion, the present study suggested that ROS and iron homeostasis dependent ferroptosis played a vital role in 5-FU induced cardiotoxicity.

Keywords: 5-FU; Cardiotoxicity; Ferroptosis; Iron homeostasis; ROS.