Abstract
Ferroptosis is a form of programmed cell death that is characterized by lipid peroxidation and is inducible by iron and the accumulation of reactive oxygen species (ROS). It is triggered by erastin but inhibited by antioxidants such as α-tocopherol, β-carotene, polyphenols, and iron chelators such as deferoxamine (DFO), nitrilotriacetic acid (NTA), and ethylenediaminetetraacetic acid (EDTA). This study investigated the protective effects of two polyphenols, curcumin and (−)- epigallocatechin-3-gallate (EGCG), against iron loading and erastin-mediated ferroptosis in MIN6 cells. Cells were treated with polyphenols before exposure to iron-induced oxidative stress comprising of 20 μmol/L of 8-hydroxyquinoline (8HQ) and 50 μmol/L of ferric ammonium citrate, (FAC) (8HQ+FAC) or Fenton reaction substrate (FS) (30 μmol/L of FeSO4 and 0.5 of mmol/L H2O2) and 20 μmol/L erastin. Cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay, iron levels were measured by inductively-coupled plasma mass spectrometry (ICP-MS), glutathione and lipid peroxidation were assayed with commercially-available kits. Curcumin and EGCG both significantly protected pancreatic cells against iron-induced oxidative damage. Moreover, both compounds also protected against erastin-induced ferroptosis in pancreatic cells. The polyphenols enhanced cell viability in erastin-treated MIN6 cells in a dose- and time-dependent manner. Furthermore, MIN6 cells exposed to erastin alone showed elevated levels of iron, glutathione (GSH) depletion, glutathione peroxidase 4 (GPX4) degradation and lipid peroxidation (p < 0.05) compared to cells that were protected by pre-treatment with curcumin or EGCG. Taken together, the data identify curcumin and EGCG as novel ferroptosis inhibitors, which might exert their protective effects by acting as iron chelators and preventing GSH depletion, GPX4 inactivation, and lipid peroxidation in MIN6 cells. The implications of the findings on the effects of iron overload and ferroptosis represent a potential therapeutic strategy against iron-related diseases.
Highlights
Iron is a vital trace metal in physiological processes such as oxygen transport, respiration, energy generation and DNA synthesis, and is a structural component of numerous enzymes and proteins in the body
In cells pre-incubated with six different polyphenols before exposure to Fenton reaction substrate (FS) and 8HQ+FAC for 2 h, only curcumin and EGCG showed significant protection against iron-induced cell damage (Figure 1A,B)
Having identified curcumin and EGCG as the most potent polyphenols for conferring protection against iron-induced stress, we investigated the potential of these compounds to act as inhibitors of ferroptosis
Summary
Iron is a vital trace metal in physiological processes such as oxygen transport, respiration, energy generation and DNA synthesis, and is a structural component of numerous enzymes and proteins in the body. Iron can exchange single electrons with metabolites to generate reactive oxygen species (ROS) that are capable of causing DNA damage, protein denaturation, and lipid peroxidation [1]. Together, these events have recently been defined as a form of programmed cell death called ferroptosis [2]. Ferroptosis emerged recently as a phenomenon that encapsulates the interplay of iron accumulation, excessive ROS levels and lipid peroxidation, and culminates in cell death that is associated with degenerative disorders [14]. The investigation was designed to demonstrate the inhibition of erastin-induced ferroptosis, iron accumulation, and lipid peroxidation by curcumin and EGCG inclusion in treated MIN6 pancreatic cells
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