Ferroptosis: Molecular Mechanisms of Regulation and Therapeutic Potential in the Pathogenesis of Multifactorial Diseases

1. Introduction

Regulated cell death (RCD) is a fundamental biological process required for tissue homeostasis, embryonic development, and immune defense [1]. For decades, apoptosis was considered the main form of programmed cell death, but the discovery of alternative mechanisms such as necroptosis, pyroptosis, and ferroptosis has significantly expanded our understanding of the pathophysiological processes [2,3]. Ferroptosis, first characterized by Dixon et al. in 2012, is an iron-dependent form of non-apoptotic cell death induced by the accumulation of lipid peroxides in membranes and characterized by unique morphological, biochemical, and genetic features [4,5]. Unlike apoptosis, ferroptosis is not accompanied by caspase activation, and its morphological marker is the decrease in mitochondrial volume with increased membrane density and the disappearance of cristae [4,6].

Over the past decade, compelling evidence has accumulated that ferroptosis plays a key role in the pathogenesis of a wide range of diseases, including malignancies, neurodegenerative disorders, cardiovascular pathology, metabolic syndromes, and inflammatory bowel diseases [7,8,9,10]. Of particular interest is the dual role of this process: induction of ferroptosis is considered a promising strategy to overcome tumor resistance to chemotherapy and immunotherapy, while its inhibition may protect tissues from ischemia-reperfusion injury and neurodegeneration [11,12,13].

Ferroptosis is a universal, complexly regulated mechanism of cell death, the understanding of which opens up new therapeutic horizons. The key challenge remains the search for specific modulators capable of differentially influencing this process depending on the pathological context, which requires in-depth study of molecular regulatory networks, in particular iron metabolism, lipids and antioxidant systems.

2. Main Text

Molecular mechanisms of ferroptosis regulation. Iron metabolism as a driving force of ferroptosis. Iron is a critical trace element required for numerous physiological processes, including oxygen transport, DNA synthesis, and mitochondrial respiration [14]. However, excess iron, especially in the form of Fe²⁺, can catalyze the Fenton reaction, generating highly reactive hydroxyl radicals that initiate lipid peroxidation [15]. It is this mechanism that underlies the iron-dependent nature of ferroptosis. Intracellular iron homeostasis is regulated by a complex network of transport proteins: transferrin (TF) binds Fe³⁺ and delivers it to the cell via transferrin receptor 1 (TFR1), after which Fe³⁺ is reduced to Fe²⁺ by STEAP3 and released into the cytoplasm via DMT1 [16,17]. Excess iron is stored in ferritin (FTH1/FTL), and its excretion is ensured by a single exporter – ferroportin (FPN) [18]. An important regulatory mechanism is NCOA4-mediated ferritinophagy – a selective autophagic process that ensures the degradation of ferritin in lysosomes with the release of free iron, which increases the sensitivity of cells to ferroptosis [19,20]. Inhibition of NCOA4 or blocking its interaction with FTH1, as shown in the study by Fang et al., significantly reduces the level of intracellular iron and prevents ferroptosis [21].

Lipid peroxidation: a central event in ferroptosis. The accumulation of lipid hydroperoxides is a key event determining the development of ferroptosis. Of particular note are polyunsaturated fatty acids (PUFA), in particular arachidonic (AA) and adrenic (AdA) acids, which are highly sensitive to oxidation due to the presence of bis-allyl hydrogen atoms [22,23]. The key enzymes in this process are ACSL4 (acyl-CoA synthetase of long-chain fatty acids) and LPCAT3 (lysophosphatidylcholine acyltransferase). ACSL4 catalyzes the esterification of free PUFA to form PUFA-CoA, which is then incorporated into membrane phosphatidylethanolamines (PE) by LPCAT3, forming PUFA-PE [24,25]. Further oxidation of PUFA-PE occurs by iron-containing lipoxygenases (LOX) or by nonenzymatic autoxidation initiated by the Fenton reaction [26,27]. Doll et al. demonstrated that ACSL4 knockout renders cells resistant to ferroptosis induced by the GPX4 inhibitor RSL3, confirming the central role of this enzyme [24]. Interestingly, recent studies have identified an alternative peroxidation pathway mediated by POR (cytochrome P450 oxidoreductase), which acts independently of LOX [28].

Antioxidant defense systems against ferroptosis. GPX4/GSH-dependent system. The primary defense mechanism against ferroptosis is the selenium-containing enzyme glutathione peroxidase 4 (GPX4), which reduces toxic lipid hydroperoxides to harmless lipid alcohols using glutathione (GSH) as a cofactor [29,30]. GSH synthesis is limited by the availability of cysteine, which enters the cell through the System Xc^- antiporter (SLC7A11/SLC3A2) in exchange for glutamate [31]. Inhibition of System Xc^- with erastin or sulfasalazine, or direct inhibition of GPX4 RSL3, leads to GSH depletion, accumulation of lipid peroxides, and ferroptosis [4,32]. Importantly, GPX4 activity depends on the incorporation of selenium in the form of selenocysteine, therefore selenium deficiency increases susceptibility to ferroptosis (Figure 1) [33].

GPX4-independent systems. In 2019, two independent groups of researchers identified FSP1 (ferroptosis suppressor protein 1, also known as AIFM2) as an alternative defense mechanism. FSP1, localized at the plasma membrane, reduces coenzyme Q₁₀ (CoQ₁₀) to ubiquinol (CoQ₁₀H₂) using NADPH, which acts as a lipophilic radical-scavenging antioxidant, inhibiting lipid peroxidation independently of GPX4 and GSH [34,35]. Doll et al. showed that expression of FSP1 protects cells from ferroptosis even in the absence of GPX4, making this pathway a promising target for therapeutic intervention [34]. Another GPX4-independent system is the GCH1-BH4-DHFR pathway. GTP cyclohydrolase 1 (GCH1) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), which has been shown to act as a potent antioxidant that prevents lipid peroxidation [36]. Soula et al. demonstrated that activation of GCH1 significantly increases the resistance of cells to ferroptosis [37]. In addition, the mitochondrial enzyme DHODH (dihydroorotate dehydrogenase) is able to reduce CoQ₁₀ to CoQ₁₀H₂ in the inner mitochondrial membrane, providing additional protection against ferroptosis at the level of this organelle (Figure 2) [38].

The role of ferroptosis in the pathogenesis of diseases. Ferroptosis in cancer therapy: opportunities and challenges. Resistance to apoptosis is one of the key characteristics of malignant cells, therefore, the induction of alternative death pathways, in particular ferroptosis, is considered a promising strategy to overcome chemoresistance [11,39]. Mesenchymal and undifferentiated cancer cells, which usually exhibit resistance to apoptosis, have been shown to be extremely sensitive to ferroptosis, making it a particularly attractive target for the therapy of resistant tumors (Figure 3) [40,41].

Synergy with immunotherapy. An important discovery was that activated CD8⁺ T lymphocytes secrete interferon-γ (IFNγ), which suppresses the expression of SLC7A11 and SLC3A2 in tumor cells through activation of the JAK/STAT1 pathway, thereby sensitizing them to ferroptosis [12]. Wang et al. showed that the combination of immunotherapy with immune checkpoint inhibitors and ferroptosis inducers (e.g., cysteinase) leads to synergistic inhibition of tumor growth in vivo [12]. Moreover, Liao et al. found that IFNγ, together with arachidonic acid, induces ferroptosis through transcriptional activation of ACSL4, opening new possibilities for combination therapy [42]. However, ferroptosis can also contribute to immunosuppression. For example, ferroptotic tumor cells release the oncogenic protein KRASG12D, which is taken up by macrophages and induces their polarization towards a pro-tumorigenic M2 phenotype through an AGER-dependent mechanism [43]. This highlights the need for a balanced approach to the modulation of ferroptosis in immunotherapy.

Synergy with radiotherapy. Radiotherapy induces ferroptosis through two main mechanisms: first, the generation of reactive oxygen species (ROS) from water radiolysis, which directly initiates lipid peroxidation; and second, the inhibition of SLC7A11 through the ATM-dependent pathway, which leads to GSH depletion [13,44]. Lei et al. demonstrated that the combination of SLC7A11 inhibitors (e.g., sulfasalazine) with radiotherapy significantly enhanced ferroptotic cell death in non-small cell lung cancer and inhibited the growth of xenografts [13]. Interestingly, hypoxia, which often accompanies solid tumors, can increase SLC7A11 expression through HIF-1α, contributing to radioresistance, making inhibition of this pathway critical for improving the efficacy of radiotherapy [45].

Controversies: The pro-tumorigenic role of ferroptosis. Despite its antitumor potential, ferroptosis may also contribute to carcinogenesis. In models of pancreatic cancer and hepatocellular carcinoma, chronic ferroptotic injury induces inflammation mediated by the release of DAMP molecules (such as HMGB1 and 8-hydroxyguanosine), which contributes to immunosuppression and tumor progression [46,47]. Dai et al. have shown that ferroptotic injury in pancreatic cells activates a STING-dependent pathway, leading to the recruitment of M2-polarized macrophages and stimulation of tumor growth [46]. Thus, the temporal and localized nature of ferroptosis induction is critical to avoid unwanted pro-inflammatory effects.

Ferroptosis in neurodegenerative and ischemic diseases. In contrast to its role in cancer therapy, the inhibition of ferroptosis serves as a protective strategy in neurodegenerative diseases and ischemic-reperfusion injuries. Iron accumulation and increased lipid peroxidation are common pathogenetic features of Alzheimer’s, Parkinson’s and Huntington’s diseases [48]. In Alzheimer’s disease, FPN expression is reduced in brain tissues. Its reduction prevents the excretion of Fe²⁺/Fe³⁺, which causes intracellular accumulation and iron overload. This leads to the induction of ferroptosis, which can be inhibited by Ferrostatin-1 [49]. In models of Parkinson’s disease, α-synuclein increases the sensitivity to ferroptosis by modulating the composition of membrane ether phospholipids [50]. Importantly, iron-chelating agents (deferoxamine, deferiprone) and lipophilic antioxidants have shown efficacy in preclinical models of neurodegeneration, and clinical trials of deferiprone in Parkinson’s disease have shown a slowing of disease progression [51,52]. In ischemic stroke, the hypoxia-inducible factor HIF-1α upregulates TFR1 expression, promoting neuronal iron accumulation and ferroptosis [53]. Inhibition of ACSL4 or activation of Nrf2 has been shown to reduce infarct size and improve neurological deficits in animal models [54,55].

Ferroptosis in cardiovascular disease. Cardiomyocytes are particularly sensitive to iron-dependent oxidative stress. Doxorubicin-induced cardiomyopathy is a classic example of ferroptosis-mediated damage: the drug forms a complex with Fe²⁺ in mitochondria, triggering lipid peroxidation and ferroptosis [56]. Tadokoro et al. demonstrated that overexpression of GPX4 in cardiomyocytes prevents this effect [56].

Atherosclerosis is also closely linked to ferroptosis: oxidized low-density lipoproteins (oxLDL) induce ferroptosis of macrophages, which contributes to the destabilization of atherosclerotic plaques [57,58]. Bai et al. showed that inhibition of ferroptosis by Ferrostatin-1 reduced lipid peroxidation and endothelial dysfunction in aortic endothelial cells of ApoE^-/- (Figure 4) [58].

Ferroptosis in reproductive and lung diseases. In the respiratory system, ferroptosis is a key trigger of acute lung injury (ALI), where lipopolysaccharide (LPS) induces iron-dependent death of alveolar epithelium, and is also involved in the pathogenesis of bronchial asthma through ALOX15-mediated peroxidation of membrane lipids and maintenance of chronic inflammation [5,23,26]. In the reproductive system, labile iron accumulation and ferroptosis of ovarian granulosa cells are critical links in the disruption of folliculogenesis and steroidogenesis in polycystic ovary syndrome (PCOS) and premature ovarian insufficiency (POI) [14,15]. Furthermore, in endometriosis, peritoneal iron overload induces ferroptosis of stromal cells with subsequent release of pro-inflammatory DAMP molecules, confirming the importance of pharmacological control of lipid peroxidation in respiratory and reproductive medicine [6,46].

Therapeutic strategies for ferroptosis modulation. Inducers of ferroptosis. Classical inducers of ferroptosis are erastin and RSL3, which inhibit System Xc^- and GPX4, respectively [4,32].

However, the clinical application of these compounds is limited by their low solubility and metabolic instability. More stable analogs have been developed, including the imidazole ketone erastin (IKE), which has shown antitumor activity in mouse models of lymphoma [59]. In addition, some FDA-approved drugs, such as sulfasalazine (an anti-inflammatory) and sorafenib (a kinase inhibitor), also induce ferroptosis in certain tumor types [60,61]. Artesunate, an artemisinin derivative, induces ferroptosis by enhancing ferritinophagy and increasing free iron levels, and has been shown to be effective against pancreatic tumors [62]. Simvastatin and other statins induce ferroptosis in triple-negative breast cancer by inhibiting the mevalonate pathway and reducing CoQ₁₀ levels [ 63].

Inhibitors of ferroptosis. The most studied inhibitors are Ferrostatin-1 and Liproxstatin-1, which act as lipid peroxyl radical scavengers, interrupting the lipid peroxidation chain reaction [4,64]. These compounds have shown efficacy in models of ischemia-reperfusion injury in the kidney, liver, and heart [65,66]. In addition, iron chelators such as deferoxamine, deferiprone, and deferasirox are widely used to treat iron overload and are being studied as potential agents for inhibiting ferroptosis in neurodegenerative diseases [67,68]. Another promising direction is the use of antioxidants such as N-acetylcysteine (NAC) and vitamin E, which increase GSH levels or directly neutralize free radicals [69].

Promising approaches: PROTACs and nanotechnology. In recent years, the development of proteolysis-targeting chimeras (PROTACs) for GPX4 degradation has received considerable attention. Luo et al. created a PROTAC-degrader of dGPX4 that selectively induces ferroptosis in cancer cells without systemic toxicity (Figure 5) [70].

Nanoparticles also open up new possibilities for targeted delivery of ferroptosis inducers. For example, nanoparticles containing RSL3 or erastin allow for high local drug concentrations in the tumor, minimizing systemic side effects [71,72].

3. Conclusions

Ferroptosis is a unique form of regulated cell death that plays a dual role in the pathogenesis of multifactorial diseases. On the one hand, induction of ferroptosis is a promising strategy to overcome the resistance of cancer cells to conventional therapies, especially in combination with immunotherapy and radiotherapy. On the other hand, inhibition of ferroptosis is crucial for protecting tissues from ischemia-reperfusion injury, neurodegeneration, and inflammation.

Key regulatory networks involving iron metabolism (TFR1, FPN, NCOA4-mediated ferritinophagy), lipid metabolism (ACSL4, LPCAT3, LOX), and antioxidant systems (GPX4/GSH, FSP1-CoQ₁₀, GCH1-BH4, DHODH) offer numerous potential targets for therapeutic intervention. Despite significant progress, unresolved issues remain: specific biomarkers of ferroptosis in vivo are lacking, the interaction between ferroptosis and other types of cell death is poorly understood, and most studies are limited to preclinical models. Future research should focus on identifying clinically relevant biomarkers, developing highly selective ferroptosis modulators with optimal pharmacokinetic properties, and conducting well-designed clinical trials to evaluate the efficacy and safety of ferroptosis-targeted therapies in patients. Understanding the context-dependent role of ferroptosis and its fine regulation is the key to successfully translating this knowledge into clinical practice.