Cell death is a fundamental biological process that maintains tissue health and eliminates damaged cells. While apoptosis and necrosis are well-known forms of cell death, ferroptosis represents a distinct, regulated pathway. Understanding and detecting this process is important for researchers and clinicians, which is why “ferroptosis markers” have become a significant area of study. These markers provide insights into when and how this unique cellular demise occurs.
The Nature of Ferroptosis
Ferroptosis is a form of regulated cell death that relies on iron and is characterized by the accumulation of lipid reactive oxygen species (ROS). This process differs from other forms of cell death, such as apoptosis, which involves cell shrinkage and DNA fragmentation, and necrosis, which is typically uncontrolled.
Ferroptosis begins with an imbalance in iron metabolism, leading to an increase in intracellular iron levels. This excess iron then catalyzes the formation of reactive oxygen species through the Fenton reaction. These reactive species attack polyunsaturated fatty acids within cell membranes, causing lipid peroxidation and ultimately cell membrane damage. The cell’s antioxidant defenses, particularly glutathione (GSH) and glutathione peroxidase 4 (GPX4), are often overwhelmed or depleted, allowing lipid peroxides to accumulate unchecked.
Identifying Key Ferroptosis Markers
Ferroptosis indicators fall into several categories, reflecting characteristic biochemical changes. Lipid peroxidation products are prominent markers, as they are the direct result of oxidative damage to cell membranes. Molecules like malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and lipid hydroperoxides are formed when polyunsaturated fatty acids in cell membranes are oxidized. Their accumulation signifies ongoing ferroptotic activity.
Iron accumulation is another direct marker, given ferroptosis’s iron dependency. An increased labile iron pool (loosely bound, redox-active iron) directly fuels reactive oxygen species generation, driving lipid peroxidation. Glutathione (GSH) depletion also serves as a marker, as this antioxidant neutralizes reactive oxygen species. Decreased intracellular GSH indicates a compromised antioxidant defense, a precursor to lipid peroxidation and ferroptosis.
GPX4 inhibition or inactivation is a central marker. This enzyme directly reduces lipid hydroperoxides to less harmful lipid alcohols, preventing ferroptosis. A loss of GPX4 function allows toxic lipid peroxides to build up, leading to cell death. Other protein markers include acyl-CoA synthetase long-chain family member 4 (ACSL4) upregulation, which promotes polyunsaturated fatty acid incorporation into membranes, increasing peroxidation susceptibility. Ferroptosis Suppressor Protein 1 (FSP1), also known as AIFM2, acts as a backup antioxidant system to GPX4; altered expression or function can also indicate ferroptosis.
Measuring Ferroptosis Markers
Various methods quantify ferroptosis markers. Biochemical assays measure lipid peroxidation products. For example, the thiobarbituric acid reactive substances (TBARS) assay quantifies malondialdehyde (MDA), a byproduct of lipid peroxidation. Assays also measure intracellular glutathione (GSH) and total iron content, often using colorimetric or fluorometric methods.
Immunohistochemistry and Western blot detect changes in ferroptosis-associated protein markers. These methods reveal alterations in protein expression or localization (e.g., GPX4, ACSL4, FSP1) in cell or tissue samples. Immunohistochemistry visualizes protein distribution within tissues, while Western blotting quantifies protein levels.
Microscopy and imaging techniques provide insights into ferroptosis’s morphological changes and detect specific molecules. Transmission electron microscopy (TEM) visualizes mitochondrial alterations like decreased mass, increased membrane density, and reduced cristae, hallmarks of ferroptosis. Lipid-soluble fluorescent probes (e.g., C11-BODIPY 581/591, LiperFluo) become fluorescent upon oxidation, allowing real-time lipid peroxidation monitoring via fluorescence microscopy or flow cytometry. Fluorescent indicators like Phen Green or calcein also detect changes in intracellular iron levels.
Genetic approaches monitor changes in ferroptosis-related gene expression. Quantitative polymerase chain reaction (qPCR) measures mRNA levels of genes like ACSL4, TFRC (Transferrin Receptor), or PTGS2 (Prostaglandin-Endoperoxide Synthase 2), whose expression often changes during ferroptosis. Increased activation of the NFE2L2 (NRF2) pathway, which regulates antioxidant genes, can also indicate oxidative stress linked to ferroptosis.
Clinical and Research Significance of Ferroptosis Markers
Ferroptosis markers have important implications for disease diagnosis and prognosis. Ferroptosis is implicated in various diseases, including certain cancers, neurodegenerative disorders (e.g., Alzheimer’s, Parkinson’s), acute kidney injury, and cardiovascular diseases. Elevated iron and lipid peroxidation markers in Alzheimer’s and Parkinson’s brains suggest ferroptosis contributes to neuronal loss. In acute kidney injury, GPX4 deficiencies can sensitize kidneys to ferroptosis.
Markers are also important in drug discovery and therapeutic development. Measuring ferroptosis markers helps identify and evaluate compounds that induce or inhibit ferroptosis. For instance, inducing ferroptosis in tumor cells is a strategy to overcome resistance to conventional cancer therapies, especially in difficult-to-treat cancers like pancreatic cancer, triple-negative breast cancer, and glioblastoma. Conversely, inhibiting ferroptosis can protect healthy cells in neurodegenerative diseases or acute organ damage.
Ferroptosis markers also hold importance in basic research. They serve as tools to unravel the mechanisms underlying ferroptosis and its physiological roles. Researchers use these markers to understand how different cellular pathways intersect with ferroptosis, how it is regulated, and its contributions to various biological processes beyond disease states. This fundamental understanding can pave the way for developing more targeted and effective therapeutic interventions in the future.