The role of estrogen and its receptors in the regulation of ferroptosis and autophagy.

Ferroptosis and autophagy are closely interconnected cellular processes, both modulated by estrogen and its receptors (ERα, ERβ, and GPER). ER signaling can inhibit ferroptosis by enhancing antioxidant defenses through NRF2 stabilization, GPX4 upregulation, and regulation of iron metabolism, or promote ferroptosis by activating NOX1 and increasing ROS production. Concurrently, ERα can induce autophagy independently of classical pathways (mTOR, PI3KCIII, MAPK) via regulators such as BAG3, ATG7, and WIPI, while ERβ modulates autophagy in a context-dependent manner, influencing autolysosome formation, cyclin degradation, HIF function, and interacting with autophagy-related proteins (ULK1, BECN1, LC3B, ATG16L1, p62) and the NLRP6 inflammasome. Selective mitophagy, promoted by compounds such as genistein, maintains mitochondrial quality and activates antioxidative regulators (SIRT3, PGC1α), modulating susceptibility to ferroptosis. These mechanisms illustrate a coordinated network in which ERs regulate both ferroptosis and autophagy, with feedback between oxidative stress, mitochondrial function, and cell death pathways. Modulation of ER signaling has important implications for cardiovascular health, neuroprotection, and cancer therapy. It may enhance ferroptosis-based anticancer strategies, influence therapy response depending on ER status, and, through compounds like phytoestrogens, provide avenues for selectively controlling ferroptotic and autophagic processes in both healthy and diseased cells. A deeper understanding of how ERs coordinate ferroptosis and autophagy will be crucial for developing precise, patient-specific therapies for cancer, cardiovascular diseases, neurodegeneration, metabolic disorders and other ferroptosis-related conditions. ER-targeted strategies thus represent a promising approach for therapeutic intervention, paving the way toward more effective and targeted medical solutions. An overview of effects of estrogen and its receptors on the regulations of ferroptosis and autophagy and their relevance to various diseases (including neurodegenerative, cardiovascular, and cancer diseases) is presented in Fig.  5 . One should note that due to differences in abundance of estrogen and its receptors between males and females, their influence on the ferroptotic and autophagic processes, and thus, their contribution to modulation of the course of various diseases might be sex-related. Fig. 5 An overview of effects of estrogen and its receptors on the regulations of ferroptosis and autophagy and their relevance to various diseases. Estrogen signaling through its receptors (acting by either stimulation of nuclear transcription factors or genome-independent, rapid pathways) is depicted in the left panel. This signaling influences ferroptosis and autophagy through different mediators (the middle panel), resulting in significant modulation of processes capable of affecting various diseases, including neurodegeneration, cancer, and cardiovascular diseases (the right panel). In the right panel, stimulation and inhibition of specific factors or processes is indicated by up arrows and down arrows, respectively An overview of effects of estrogen and its receptors on the regulations of ferroptosis and autophagy and their relevance to various diseases. Estrogen signaling through its receptors (acting by either stimulation of nuclear transcription factors or genome-independent, rapid pathways) is depicted in the left panel. This signaling influences ferroptosis and autophagy through different mediators (the middle panel), resulting in significant modulation of processes capable of affecting various diseases, including neurodegeneration, cancer, and cardiovascular diseases (the right panel). In the right panel, stimulation and inhibition of specific factors or processes is indicated by up arrows and down arrows, respectively

Iron circulates in two forms in the organism, ferrous (Fe²⁺) and ferric (Fe³⁺), both of which are crucial for maintaining cellular homeostasis [ 9 ]. Both iron deficiency and its excess may lead to disturbances in fundamental metabolic processes, highlighting the need for tight regulation within tissues [ 10 ]. Iron metabolism, which is defined by its availability, transport, and redox state, is closely associated with ferroptosis. This process is mediated by a network of compounds that can induce ferroptotic pathways [ 2 ]. Cells acquire iron primarily in the form of Fe³⁺ ions bound to transferrin. Transferrin is a glycoprotein that reversibly binds Fe³⁺ ions and assists in their transport to the cell membrane. Once the transferrin-Fe³⁺ complex reaches the membrane, it interacts with the transferrin receptor 1 (TFR1), enabling Fe³⁺ internalization into the cell [ 11 ]. The reduction of iron to Fe²⁺ inside the cell occurs in endosomes through the action of the STEAP3 ferrireductase, which reduces iron. In its reduced state, iron is then transported from the endosome to the cytoplasm via the SLC11A2 transporter, where it is stored by binding to ferritin, constituting a stable storage form of iron. It can also remain free as part of LIP. The excess of unbound Fe²⁺ in the cytoplasm is converted to Fe³⁺ by ferroportin [ 11 , 12 ]. Free Fe²⁺ forms, not bound to ferritin and not oxidized by ferroportin, undergo the Fenton reaction. This reaction generates ROS, which are highly unstable, and propagate lipid peroxidation processes. Another compound modulating ferroptosis specifically by contributing to the accumulation of free Fe²⁺ ions, is frataxin (FXN). FXN is a mitochondrial protein involved in mediating redox reactions and regulating iron homeostasis. Studies by Du et al. have shown that suppression of expression of a gene encoding FXN disrupts the process of iron-sulfur cluster formation, resulting in the accumulation of free Fe²⁺ ions [ 13 ]. As mentioned earlier, the accumulation of excessive Fe²⁺ fuels the Fenton reaction, therefore deficiency of FXN can be considered an inducer of ferroptosis. All aforementioned iron-dependent processes leading to ferroptosis are presented in Fig.  1 . An interesting question is whether there are feedback loops between ferroptosis and iron homeostasis. Specifically, one might ask if ferroptotic cell death could modulate cellular iron levels via ferroportin-ferritin regulation. Potentially such a feedback might exits, especially as variability of the levels of ferrous ions in ferroptotic cells could be huge [ 14 ]. Thus, depending on activities of ferropotin and ferritin, such ions can either be transported outside the cell or being further more abundant inside. If the former option is true, it could perhaps contribute to the phenomenon of trigger waves, as it was demonstrated recently that ferroptosis can propagate across cells over long distances (in a range of several mm) [ 15 ]. Indeed, a ferroptotic stress was shown to be able to activate ROS feedback loops, resulting in long-distance ROS propagation [ 15 ]. Therefore, the regulatory processes of ferroptosis and iron homeostasis may be complex and reciprocal, significantly influencing cell physiology. The process of unrestrained lipid peroxidation is considered fundamental characteristics of ferroptosis-induced cell death. It involves the oxidation of lipids present in cellular membranes, leading to their damage. It encompasses chemical reactions between oxygen, PUFA and in the context of ferroptosis - iron. While PUFA and oxygen are directly involved in the reaction, iron plays a critical role in intensifying the process. Excess of Fe²⁺ ions participates in the Fenton reaction, generating ROS. ROS initiate and propagate lipid peroxidation, leading to the formation of lipid radicals and peroxides, which further damage PUFA. Consequently, iron drives the chain reaction, exacerbating membrane degradation [ 2 ]. The mechanism of lipid peroxidation begins with the attack of oxidants, such as free radicals, targeting the carbon-carbon double bonds within the PUFAs. The target of this attack is the bis-allylic group, located between two double bonds, particularly susceptible to breakage [ 1 ]. As a result, a hydrogen atom is removed from the lipid molecule, leading to the formation of a carbon-centered lipid radical, which then reacts with oxygen and propagates chain reactions that result in progressive lipid oxidation and exacerbation of membrane damage [ 10 , 16 ]. This disruption of membrane integrity serves as a pivotal factor in the mechanism of ferroptosis. Fig. 1 Regulation of ferroptosis via the iron-dependent pathway. The figure illustrates the transport of Fe³⁺ into the cell via the transferrin receptor (TFR1), where it enters the endosome and is reduced to Fe²⁺ by the STEAP3 ferrireductase. It is then transported to the cytoplasm by the SLC11A2 transporter and may remain in a ferritin-unbound form. Free Fe²⁺ ions undergo the Fenton reaction, leading to the accumulation of reactive oxygen species (ROS). ROS directly damage phospholipid structures, and this process cascades to damage cell membranes, ultimately causing ferroptosis. Created using Microsoft PowerPoint Regulation of ferroptosis via the iron-dependent pathway. The figure illustrates the transport of Fe³⁺ into the cell via the transferrin receptor (TFR1), where it enters the endosome and is reduced to Fe²⁺ by the STEAP3 ferrireductase. It is then transported to the cytoplasm by the SLC11A2 transporter and may remain in a ferritin-unbound form. Free Fe²⁺ ions undergo the Fenton reaction, leading to the accumulation of reactive oxygen species (ROS). ROS directly damage phospholipid structures, and this process cascades to damage cell membranes, ultimately causing ferroptosis. Created using Microsoft PowerPoint PUFA contain one or more polyunsaturated fatty acyl tails and are the most susceptible to peroxidation. When the production of PUFA increases within a cell, it becomes more vulnerable to ferroptosis. Different enzymes play a pivotal role in both the synthesis and peroxidation of PUFA, driving their metabolic integration. Acyl-CoA synthetase long-chain family member 4 (ACSL4) serves as a positive regulator of ferroptosis [ 17 ]. It converts PUFA into bioactive intermediates that can be used in biochemical processes. ACSL4 esterifies PUFA into an acyl-CoA, resulting in the formation of PUFA-CoA. These activated PUFA derivatives are then incorporated into cellular phospholipids (PL) by lysophosphatidylcholine acyltransferase 3 (LPCAT3), generating PL-PUFA complexes [ 2 , 10 ]. Importantly, PUFA composition varies across tissues, influencing ferroptosis sensitivity [ 18 ]. This susceptibility depends not only on total PUFA levels but also on the specific lipidomic signature, their incorporation into membrane phospholipids, and tissue-specific lipid remodeling and antioxidant capacity [ 17 , 19 ]. For instance, the brain is enriched in specific highly peroxidizable long-chain PUFA such as DHA, increasing vulnerability to ferroptosis. Both ω‑3 and ω‑6 PUFA, due to multiple double bonds, are particularly susceptible to lipid peroxidation, whereas tissues in other organs, such as liver or skeletal muscles, exhibit different PUFA profiles and remodeling phenotypes that may confer relative resistance to ferroptosis [ 18 , 20 ]. In disease contexts, such tissue-specific PUFA profiles modulate ferroptotic outcomes, contributing to neurodegeneration, atherosclerosis or influencing cancer cell survival and therapeutic response [ 21 – 24 ]. The regulation of ferroptosis is strongly dependent on GPX4, a central enzyme responsible for protecting cells from oxidative damage by neutralizing lipid peroxides. GPX4 plays a pivotal role in maintaining cellular membrane integrity by reducing toxic lipid hydroperoxides (L-OOH) to their corresponding alcohols (L-OH), with GSH serving as a cofactor. This enzymatic activity halts the propagation of lipid peroxidation, a process central to ferroptosis [ 1 ]. The cystine/glutamate antiporter system Xc⁻, composed of the light chain subunit xCT (encoded by the SLC7A11 gene) and the heavy chain subunit 4F2hc (encoded by SLC3A2 ), is a critical regulator of ferroptosis. xCT is the functional subunit responsible for the actual transport activity, and its presence is commonly used as a surrogate marker for system Xc⁻ function. This transporter imports cystine into cells, which is essential for GSH biosynthesis. As GSH is required for the activity of GPX4, xCT indirectly protects cells from ferroptosis by maintaining intracellular antioxidant defenses [ 16 ]. Inhibition of xCT reduces cystine uptake, depleting GSH levels and impairing GPX4 function. This relationship between xCT, GSH synthesis, and GPX4 activity is visually summarized in Fig.  2 , which outlines the regulatory pathway preventing ferroptosis. Fig. 2 Regulation of ferroptosis via the GSH/Xc-/GPX4 pathway. The cystine/glutamate antiporter (Xc⁻) facilitates the uptake of cysteine, which is essential for glutathione (GSH) synthesis. GSH acts as a cofactor for GPX4, an enzyme responsible for neutralizing lipid peroxides. When Xc⁻ is inhibited or cystine availability is reduced, GSH levels decrease, impairing GPX4 activity. This leads to the accumulation of lipid peroxides and reactive oxygen species (ROS), ultimately triggering ferroptosis. Additionally, factors such as p53 and Beclin-1 can downregulate Xc⁻, further promoting ferroptotic processes. Created using Microsoft PowerPoint Regulation of ferroptosis via the GSH/Xc-/GPX4 pathway. The cystine/glutamate antiporter (Xc⁻) facilitates the uptake of cysteine, which is essential for glutathione (GSH) synthesis. GSH acts as a cofactor for GPX4, an enzyme responsible for neutralizing lipid peroxides. When Xc⁻ is inhibited or cystine availability is reduced, GSH levels decrease, impairing GPX4 activity. This leads to the accumulation of lipid peroxides and reactive oxygen species (ROS), ultimately triggering ferroptosis. Additionally, factors such as p53 and Beclin-1 can downregulate Xc⁻, further promoting ferroptotic processes. Created using Microsoft PowerPoint The inactivation of GPX4 has been shown to sensitize cells to ferroptosis, underscoring its importance as a critical checkpoint in this cell death pathway. Disrupting GPX4 function leads to the accumulation of lipid peroxides, ultimately compromising membrane integrity and triggering ferroptosis [ 1 ]. As a result, targeting GPX4 has become a strategic focus in ferroptosis research, particularly for developing therapies to combat treatment-resistant cancers and diseases linked to oxidative stress. A variety of inhibitors have been identified that act on GPX4 directly or indirectly, disrupting its function and inducing ferroptosis. These inhibitors are invaluable tools for understanding ferroptosis mechanisms and hold therapeutic potential for manipulating this pathway in disease contexts. In fact, the therapeutic potential of small molecules modulating GPX4 activity has been discussed recently, indicating that they might be considered as promising drug candidates for treatment of different diseases, including cancers, neurodegenerative diseases, inflammatory disorders, ischemia/reperfusion injury, a specific type of spondylometaphyseal dysplasia, and even some infectious diseases, like tuberculosis [ 25 ]. On the other hand, it was demonstrated that although downregulation of expression of a gene coding for GPX4 could induce the ferroptosis process in multiple adherent cancer cell cultures, the efficacy of this approach in animal and 3D cellular models was reduced due to ferroptosis resistance, caused by enhanced expression of a gene encoding stearoyl-CoA desaturase, and resultant altered ratio of monounsaturated fatty acids (MUFAs) to PUFAs [ 26 ]. Therefore, despite a GPX4 inhibitors are promising therapeutic agents, it is crucial to consider physiological conditions which might modulate their efficacy in the organism. FSP1 (previously named apoptosis-inducing factor mitochondria associated 2, AIFM2), is an inhibitor of ferroptosis [ 27 ]. This NADP-dependent oxidoreductase protects cellular lipids from oxidative stress, preventing peroxidation and maintaining membrane integrity. FSP1 is predominantly localized in the plasma membrane, where the presence of an N-terminal myristylation motif facilitates its anchoring. The significance of this motif was substantiated by studies which demonstrated that mutations within the myristylation site (G2A) of FSP1 negatively affected its ferroptosis inhibition activity [ 28 ]. The ferroptosis regulatory pathways associate with FSP1 function independently of the glutathione-glutathione peroxidase 4 (GSH-GPX4) pathway [ 16 ]. FSP1 regulates ferroptosis through several distinct metabolic pathways, notably the FSP1-CoQ10-NAD(P)H axis. This system involves the conversion of CoQ10 (the oxidized form of coenzyme Q10, also known as ubiquinone) into its reduced form, CoQ10H2 (ubiquinol), with the help of NAD(P)H [ 9 ]. CoQ10 serves as a lipophilic electron carrier in the mitochondrial respiratory chain, while CoQ10H2 acts as a potent antioxidant, scavenging free radicals. By reducing lipid peroxidation, CoQ10H2 helps to protect cells from oxidative stress. Thus, FSP1 operates independently of GPX4, with both FSP1 and GPX4 working together as complementary defense mechanisms against ferroptosis [ 1 , 16 ]. The estrogen signaling pathway is another regulatory process which ensures cellular redox homeostasis. In fact, as discussed recently, in the absence of estrogen receptor, the FSP1-CoQ10-NAD(P)H axis was proposed to be a key process ensuring the maintenance of this homeostasis, enabling cell survival [ 26 ]. Therefore, there is a clear connection between ferroptosis and estrogen functions, which will be discussed in more detail in subsequent sections of this paper. It appears that this interplay between estrogen-dependent control of metabolism and ferroptosis (and autophagy) might be especially important under physiological stress conditions, thus influencing various pathological processes and the course of diseases. Estrogen is a group of steroid hormones that regulate biological processes in both women and men by activating endogenous estrogen receptors (ERs). The main endogenous estrogens in humans − 17β-estradiol (E2), estrone (E1), and estriol (E3) - differ in potency, while estetrol (E4), produced during pregnancy, has recently gained interest [ 29 ]. The content of a specific form of estrogen in the organism is presented in Fig.  3 , along with the processes in which estrogen is involved [ 30 ]. Estrogen levels fluctuate in women, with a sharp decline during menopause from 100 to 250 pg/ml to ~ 10 pg/ml (while in men, they remain more stable 10–40 pg/ml). The sharp decline in estrogen levels during menopause reveals the protective roles these hormones had previously played in various bodily systems [ 31 ]. Estrogens are primarily synthesized in the gonads but are also produced in extragonadal tissues such as the brain, adipose tissue, bone, liver, and blood vessels. In the ovaries, estrogen synthesis begins in theca cells with androgen production and is completed in granulosa cells via aromatase. In men, estrogens are synthesized in Leydig and Sertoli cells, as well as in mature spermatocytes. While extragonadal tissues cannot produce C19 steroid precursors, they convert them into estrogens through aromatase activity, relying on external precursor supply. These estrogens act locally and are metabolized within their site of production, limiting systemic effects. Understanding extragonadal estrogen synthesis is crucial for developing therapeutic strategies targeting estrogen signaling in disease prevention and treatment. Since estrogen action is mediated by its receptors, tissues expressing one or more ERs are considered key targets for regulation [ 32 ]. Fig. 3 Specific forms of estrogen in the body and their involvement in various disorders and diseases, with consideration of their relative abundance. These endogenous estrogens differ in potency and biological roles, with E4 being produced during pregnancy. E1 – estrone, E2–17β-estradiol, E3 – estriol, E4 – estetrol. Created with BioRender.com Specific forms of estrogen in the body and their involvement in various disorders and diseases, with consideration of their relative abundance. These endogenous estrogens differ in potency and biological roles, with E4 being produced during pregnancy. E1 – estrone, E2–17β-estradiol, E3 – estriol, E4 – estetrol. Created with BioRender.com Estrogen also exerts a significant influence on cancers, particularly breast cancer. Depending on the tumor type, disease context, and the mode of estrogen administration, it can either promote or inhibit carcinogenesis. In ER+ cancers, combining anti-estrogen therapy with CDK4/6 inhibitors (e.g., palbociclib) enhances treatment by arresting the cell cycle and increasing susceptibility to oxidative stress. Blocking ERs weakens cellular defense against ferroptosis. Inhibiting GPX4, which neutralizes lipid peroxidation, sensitizes ER+ breast cancer cells to palbociclib and giredestrant, improving therapy effectiveness. This suggests ferroptosis as a potential therapeutic target in ER+ breast cancer( 33 ). Some studies indicated that estrogen and excessive phytoestrogen intake can promote breast cancer cell proliferation. Importantly, it was indicated that not all forms of estrogen exposure carry the same risk, as vaginal estrogen therapy has been shown to be safe and not increasing mortality. However, selective antagonism of estrogen signaling remains one of the most effective treatment approaches [ 34 ]. Estrogen broadly influences the autophagy process through both endogenous and exogenous ligands of ERs. Since estrogen balances the expression of autophagy-related genes, this regulation can be both stimulatory and inhibitory [ 35 ]. Cells producing ERα exhibit higher autophagic activity than cells producing ERβ and cells unable to synthesize lacking ERα [ 36 ]. It has been demonstrated that estrogen contributes to promoting autophagy by increasing the production of LC3-II as a result of hypertension induced by hypoxia [ 37 ]. Estrogen plays a significant role in the regulation of energy metabolism, particularly by modulating the activity of proteins and receptors involved in fat metabolism. Following the loss of estrogen, what occurs during menopause, the body’s energy efficiency decreases, which can lead to obesity and other metabolic disorders. These changes may affect pathways associated with lipid metabolism, especially lipids that are susceptible to peroxidation. Estrogen, due to its antioxidant properties, warrants discussion in the context of lipid peroxidation( 31 ). A study conducted by Tian et al. demonstrated that efficiency of lipid peroxidation in hippocampal neurons was elevated following treatment with erastin (a compound known to induce ferroptosis), while the use of E2 tended to normalize lipid peroxidation levels again [ 38 ]. It has been reported that E2 protects against oxidative stress by promoting the production of mitochondrial antioxidant enzymes (SOD2, GPX), increasing levels of antioxidants, and reducing abundance of free radicals in various organs and cells [ 16 ]. Considering anti-inflammatory effects, estrogen has been shown to protect the blood-brain barrier (BBB) from disruption by inflammation. Furthermore, it has been suggested that this effect is mediated by the anti-inflammatory protein annexin A1 (ANXA1), which contributes to reduced barrier permeability by promoting the formation of tight junctions and enhancing the integrity of endothelial cells [ 39 ]. Particularly relevant in the context of this review is the fact that estrogen exhibits antioxidant properties, supporting the removal of ROS, which exacerbate the process of ferroptosis. The role of it in the process of ferroptosis can be complex and context-dependent, influenced by the presence, levels, or absence of its receptors. Estrogen can modulate the expression of genes associated with iron transport and storage, such as those coding for ferroportin (iron transporter), FPN1 (iron exporter), DMT1 (iron importer), iron regulatory protein (IRP1), and ferritin (iron storage protein) [ 40 ]. E2 exhibits protective effects by reducing iron content in the brain through the regulation of iron-transporting proteins, thereby potentially playing a key role in protecting against neurodegenerative diseases associated with iron accumulation, such as Alzheimer’s disease and Parkinson’s disease [ 41 ]. This also applies to rare neurodegenerative disorders such as neurodegeneration with brain iron accumulation (NBIA), which are characterized by progressive neuronal damage and pathological iron deposition, particularly in the basal ganglia. Although NBIA syndromes are genetically heterogeneous, they share iron dysregulation as a hallmark, further emphasizing the importance of iron homeostasis and its potential modulation by estrogen [ 42 ]. Estrogen deficiency under postmenopausal conditions leads to disruptions in iron metabolism and activation of signaling pathways responsible for the development of postmenopausal osteoporosis (PMOP). Low E2 levels result in iron accumulation in the skeleton through dysregulation of the Nrf2 pathway, which increases the production of RANKL (receptor activator of nuclear factor kappa-B ligand), promotes the generation of ROS, and initiates osteocyte ferroptosis by reducing the activity of GPX4 [ 43 ]. Animal model studies have demonstrated that low E2 levels lead to increased bone resorption, as observed by elevated levels of the bone resorption biomarker (β-CTX), without a compensatory increase in bone formation (osteocalcin). When iron supplementation was added, the deterioration of bone condition was further exacerbated. This suggests that iron accumulation in the body (particularly under conditions of E2 deficiency) enhances bone resorption processes, further weakening bone strength and accelerating bone mass loss [ 44 ]. Disruption of the HIF-1α/FSHR/CYP19A1 signaling pathway by excess of iron results in reduced E2 synthesis efficiency in granulosa cells, where HIF-1α (Hypoxia-inducible factor-1α) regulates the cell’s response to stress induced by excess iron and ROS, FSHR (follicle-stimulating hormone receptor) influences the maturation and function of granulosa cells, and CYP19A1 (aromatase) is the enzyme responsible for the biosynthesis of E2 from androgens. Decreased E2 levels in this context may impact oocyte maturation and fertility [ 45 ]. This confirms that low E2 levels can be both beneficial and detrimental, depending on whether ferroptosis is desirable or not in a particular disease condition. MBOAT1 is an inhibitor of ferroptosis, functioning by shifting cellular lipid remodeling toward MUFAs instead of PUFAs, thus preventing lipid peroxidation. This shift is especially critical because PUFAs are highly susceptible to peroxidation, which drives ferroptosis. MBOAT1 plays a protective role by stabilizing lipid membranes and reducing oxidative damage. Furthermore, MBOAT1 expression is regulated by the ERα, linking it to estrogen-driven pathways. Estrogen activation of ERα increases MBOAT1 expression, which may help estrogen-dependent cancer cells, such as breast cancer cells, evade ferroptosis by modulating lipid metabolism. In this way, MBOAT1, in coordination with estrogen signaling, supports cancer cell survival by stabilizing lipids and preventing ferroptosis. This concept has been presented in an illustrative and metaphorical manner [ 46 ]. Recent studies, including works on CRISPR activation screenings, have identified MBOAT1 and MBOAT2 as newly identified ferroptosis inhibitors that function independently of GPX4 or FSP1 by remodeling phospholipids [ 47 ]. Transcription of their genes is upregulated by ER and androgen receptors (AR), respectively. The induction of ferroptosis, combined with ER or AR antagonists, significantly inhibits the growth of ER-positive breast cancer or AR-positive prostate cancer, offering a new therapeutic approach for cancers with specific genetic background [ 48 ]. Proposing the use of endogenously occurring estrogen, followed by its observation and targeted regulation through ERs in the context of ferroptosis, represents an intriguing area warranting further research. ERs play a key role in regulating various complex physiological processes in humans [ 49 ]. ERs are divided into three subtypes: ERα, ERβ and GPER1 [ 50 ]. ERα and ERβ belong to the family of steroid-activated transcription factors. In contrast, GPER1 is a membrane-associated G protein-coupled receptor that operates in a distinct manner. It participates in rapid, non-genomic estrogen signaling by activating various protein kinase cascades such as protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) (cases have also been observed where GPER1 regulates the expression of specific genes, however, this is not its typical mode of action) [ 39 , 50 , 51 ]. All three ER subtypes are distinguished by their locations within cells and the organism. ERα and ERβ are typically located in the nucleus, where they act as transcription factors regulating gene expression. In their unbound form, they can also be transiently present in the cytoplasm, from where they translocate to the nucleus upon estrogen binding. GPER1 is primarily located in the plasma membrane but is also found in the endoplasmic reticulum and the Golgi apparatus [ 51 ]. Within the organism, ERα is mostly present in reproductive tissues such as ovaries and uterus, kidneys, bones, breast and liver. On the other hand, ERβ is found in the central nervous system, male reproductive organs, lungs, cardiovascular and immune system, kidneys and colon [ 49 ]. GPER1 has a broader distribution and occurs in neurons, skeletal muscles, liver, immune cells, vascular endothelium and target effector organs. Furthermore, GPER1 was reported to be present in ovarian, breast and lung cancer tissues [ 49 ]. The ER protein molecule is composed of A/B, C, D and E/F domains arranged from the amino to the carboxyl terminals. ERα and ERβ are encoded by the ESR1 gene, located in chromosome 6, and the ESR2 gene, present in chromosome 14, respectively [ 49 ], [ 40 ]. Disruptions in ER signaling are implicated in a range of disorders, including endometriosis, prostate, breast and ovarian cancers, lung cancer, as well as cardiovascular, gastrointestinal, neurodegenerative, and mental disorders [ 49 ]. ERα regulates neurobiological systems related to reproduction, such as those involved in sexual characteristics and maturation, while ERβ is implicated in the modulation of non-reproductive systems, including locomotion, memory and learning, anxiety and fear [ 40 ]. Knockout of the gene encoding ERα in female mice results in hyperphagia, obesity, hypometabolism, and insulin resistance. Similar, metabolic disturbances are observed after genetic deletion of the gene coding for the aromatase enzyme (ArKO) in both sexes. In contrast, the deficiency of ERβ has not been associated with obesity or any related metabolic changes [ 52 ]. ERα plays a crucial role in mitigating inflammatory processes, regulating visceral fat metabolism, and supporting the actions of estrogens in lowering blood pressure. Its absence in key tissues leads to heightened inflammation, metabolic disturbances, and a weakened protective effect of estrogens on the cardiovascular system [ 52 ]. ERα modulates the expression of genes involved in various cellular processes, such as proliferation, differentiation, and apoptosis, by interacting with proteins c-Fos and c-Jun, which are part of the AP-1 complex responsible for regulating these processes. Of particular interest is the antagonism between ERα and ERβ in the regulation of expression of a gene encoding cyclin D1 a key regulator of the cell cycle. While ERα induces cyclin D1 gene expression, ERβ inhibits its expression. Gene coding for the membrane receptor GPER1 is located in chromosome 7. GPER1 activation initiates multiple intracellular signaling cascades, such as the phosphoinositide 3-kinase (PI3K) pathway, mitogen-activated protein kinase (MAPK) pathways and the production of cyclic adenosine monophosphate (cAMP). These pathways influence cell proliferation, survival, and migration. Notably, GPER1 can transactivate the epidermal growth factor receptor (EGFR), leading to downstream signaling events that affect cellular functions [ 53 ]. In the context of reproductive health, GPER1 has been studied for its involvement in female reproductive cancers, including ovarian and endometrial cancers. It was suggested that GPER1 may influence tumor growth and progression, highlighting its potential as a therapeutic target [ 54 ]. GPER1 plays a significant role in cardiovascular physiology. Activation of GPER1 has been associated with vasodilation, reduced blood pressure, and protection against vascular injury. These effects are mediated through the activation of endothelial nitric oxide synthase (eNOS) and the subsequent production of nitric oxide (NO), a potent vasodilator [ 55 ]. Emerging evidence indicates that GPER1 modulates immune cell function. It has been shown to influence the activation and proliferation of T lymphocytes, B lymphocytes, and macrophages. Additionally, GPER1 may play a role in the pathogenesis of autoimmune diseases and cancer by affecting immune cell responses [ 56 ]. Estrogens regulate various metabolic processes, including iron homeostasis and lipid metabolism, through the activation of ERs. For example, they lower free iron levels by inhibiting the expression of genes encoding transferrin receptor protein 1 (TFRC) and ferritin while promoting ferroportin production. Additionally, estrogens suppress lipid peroxidation via the Wnt/β-catenin pathway, which may influence ferroptosis [ 39 ]. This section will explore the role of ERs in regulating ferroptosis and examine how specific ligands or phytoestrogens modulate receptor activity to either enhance or inhibit ferroptotic processes. Furthermore, we will discuss the implications of ER-mediated ferroptosis for the brain function, neurodegenerative diseases, hormonal disorders, inflammation, and cancer. All three ERs appear to modulate ferroptosis-related pathways through interconnected mechanisms that are still being investigated. Their overlapping roles suggest involvement in the regulation of oxidative stress and iron metabolism [ 57 ]. Figure  4 illustrates the involvement of ERs in the regulation of oxidative stress and iron metabolism, highlighting the key signaling pathways through which they may influence ferroptosis. Fig. 4 The role of estrogen receptors in regulating cellular signaling, oxidative stress, and ferroptosis. The figure illustrates the interactions between Nrf2 activation, EGFR–ERK–PI3K/Akt pathways, ROS production, and iron metabolism, highlighting the potential influence of ERs on both pro- and anti-ferroptotic processes. Created with BioRender.com The role of estrogen receptors in regulating cellular signaling, oxidative stress, and ferroptosis. The figure illustrates the interactions between Nrf2 activation, EGFR–ERK–PI3K/Akt pathways, ROS production, and iron metabolism, highlighting the potential influence of ERs on both pro- and anti-ferroptotic processes. Created with BioRender.com The “ferroptotic tone” of cells may be influenced by the nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor which regulates levels of several key components of the ferroptosis pathway. Estrogens rapidly activate NRF2 via ERs and GPER1, positioning NRF2 as a central regulator of detoxification and metabolic systems that support cell proliferation [ 58 ]. Stimulation of ERα induces transactivation of the epidermal growth factor receptor (EGFR), activating downstream Ras/RAF/MEK/ERK and Ras/PI3K/PTEN/Akt signaling pathways. Activated Akt inhibits GSK3β, stabilizing NRF2 and increasing its nuclear import. This cascade involves molecules such as HB-EGF (heparin-binding EGF-like growth factor), ADAM17, PLC (phospholipase C), PLD (phospholipase D), Prx6 (peroxiredoxin 6), NOXA1, and NOXO1 [ 58 ]. GPER1 signaling works synergistically with ERα-EGFR signaling to activate NADPH oxidase 1 (NOX1), generating superoxide anions. These anions are metabolized to H₂O₂, amplifying EGFR signaling. Activation of Rac1, PKCβ, and ERK enhances NOX1 activation. GPER1 agonist G1 also inhibits plasma membrane Ca²⁺-ATPase (PMCA) activity, increasing intracellular Ca²⁺ levels. Additionally, GPER1 upregulates NOX1 expression, enhancing ROS production and potentially promoting ferroptosis, as observed in Parkinson’s disease models [ 59 ]. In osteoarthritis (OA) models, NOX1-induced oxidative stress triggers chondrocyte ferroptosis by suppressing the NRF2/HO-1 pathway, highlighting the role of specific ER isoforms and GPER1 in NOX1 activation [ 60 ]. Keap1-Nrf2 protein-protein interaction (PPI) inhibitors show potent antioxidant effects by activating NRF2 signaling, suppressing prooxidative and proinflammatory gene expression induced by estrogen. While estrogens can increase oxidative stress by inducing these genes, Keap1-Nrf2 PPI inhibitors counteract such effects, enhancing antioxidant defenses and protecting cells from oxidative damage [ 61 ]. Recent studies demonstrated that estrogen and ERβ exhibit protective antioxidant effects, primarily by promoting FGF2 production, which protects Müller and other retinal cells from oxidative damage. Remarkably, these effects are independent of NRF2 signaling. Even after NRF2 gene deletion, estrogen prevents Müller cell death, induces FGF2 production, and protects photoreceptor cells from oxidative stress in vivo. This suggests that exogenous estrogen or selective ERβ agonists may offer therapeutic benefits for retinal degenerative diseases, particularly in postmenopausal women with reduced endogenous estrogen levels [ 62 ]. Studies on the cardioprotective effects of estrogen in women have demonstrated that both estrogen and its receptors can limit the activation of ferroptosis and ferritinophagy, a selective autophagy process mediated by nuclear receptor coactivator 4 (NCOA4), which involves the recognition of ferritin by NCOA4 and its delivery to the autophagosome. In the lysosome, ferritin is degraded, releasing iron which may contribute to the initiation of ferroptosis [ 63 ]. This process is tightly regulated by metabolic and signaling pathways such as AMPK/mTOR, AMPK, Nrf2, and p53 [ 64 ]. The p62/Keap1/Nrf2/HO-1 pathway is particulary important in regulating susceptibility to ferroptosis by influencing the generation of ROS [ 65 ]. ERs modulate these pathways, reducing oxidative stress and iron metabolism dysregulation, contributing to sex-specific resilience in conditions like Takotsubo syndrome [ 64 ]. Additionally, p53, once activated, can bind to the Slc7a11 promoter region, suppressing its transcriptional activity. This suppression reduces the cell’s antioxidant capacity and increases its susceptibility to ferroptosis, further highlighting the interplay between estrogen signaling and ferroptosis regulation [ 66 ]. In ApoE −/− mice with (OVX) and high-fat diets, mimicking postmenopausal atherosclerosis, estrogen reduces ferroptosis markers, like lipid peroxidation and iron deposition. E₂ mitigated endothelial ferroptosis induced by oxLDL or erastin through antioxidant mechanisms, improving mitochondrial function and upregulating GPX4 expression. This effect was dependent on NRF2 activation, highlighting the NRF2/GPX4 pathway as a mediator of E2’s protective role. ERs likely drive these effects by regulating antioxidant pathways, reducing oxidative stress, and protecting endothelial cells from ferroptotic damage [ 8 ]. ERs play a significant role in protecting against osteoarthritis (OA), by exhibiting anti-ferroptotic effects crucial for chondrocyte survival. Consequently, ERs may protect against joint degeneration through ferroptosis-related mechanisms, highlighting their potential as therapeutic targets for OA and other related diseases, such as mucopolysaccharidoses. These conditions share common features, including cartilage destruction, joint pain and stiffness, and joint deformities [ 67 , 68 ]. Studies were also conducted on multiple compounds that may regulate the ferroptotic process through interactions with ERs. Qixian granule (QXG), is a traditional Chinese medicine formula which consists of herbs known for their role in promoting circulation, reducing inflammation and protecting against oxidative stress. Icariin, an active ingredient in Qixian Granule, inhibits ferroptosis by reducing oxidized LDL-induced endothelial cell injury and promoting autophagy in atherosclerotic mice. Salvianolic Acid B prevents ferroptosis and apoptosis during myocardial ischemia/reperfusion injury by stabilizing GPX4 and reducing oxidative stress through the ROS-JNK/MAPK pathway. Qixian granule also inhibits ferroptosis in vascular endothelial cells, which is implicated in postmenopausal atherosclerosis. The granule modulates TRPML1 in the lysosome, potentially protecting endothelial cells from ferroptosis-related damage. Estrogenic components in QXG may contribute to this protective effect, although the precise role of ERs in regulating ferroptosis in this context requires further investigation [ 69 ]. Activation of GPER1 by bisphenol A (BPA) disrupts lipid metabolism and induces ferroptosis in the liver, linking GPER1- mediated estrogenic signaling to oxidative stress and ferroptotic cell death. In chicken models, BPA exposure altered the expression of genes involved in fatty acid β-oxidation, lipid synthesis and uptake. This was accompanied by a significant increase in pro-inflammatory cytokines, including IL-1β, IL-18, and TNF-α. BPA also promoted hepatic ferroptosis by increasing iron accumulation and upregulating genes associated with lipid peroxidation, while downregulating key antioxidant-related genes such as GPX4 , SLC7A11 , and SLC3A2 . Notably, inhibiton of GPER1 significantly attenuated BPA-induced lipid metabolic disturbances, inflammatory responses, and ferroptosis underscoring the central role of GPER1 in mediating BPA-induced hepatic dysfunction [ 70 ]. These findings highlight the involvement of GPER1 in ferroptosis during BPA-induced hepatotxicity, providing new insights into the risks of BPA exposure and establishing GPER1 as a critical regulator of ferroptotic pathways. Daidzein (DA) is released from the diet by the enzyme β-galactosidase produced by intestinal bacteria, specifically by a strain of Lactobacillus vaginalis . This enzyme converts isoflavone glycosides present in the food into their aglycone forms, such as DA, which have better bioavailability and antioxidant properties [ 71 ]. DA has been shown to mitigate acetaminophen-induced hepatotoxicity in mice. This compound, through its estrogen-like activity, may play a role in modulating ferroptosis pathways, offering protective effects against liver damage by influencing oxidative stress and iron homeostasis [ 71 ]. Similarly, in endometriosis, E 2 upregulates ERα and ERβ, inhibiting ferroptosis via the LCN2 pathway, contributing to endometriotic tissue survival [ 72 ]. E2 increases efficiency of the LCN2 gene expression in a dose-dependent manner in human adipose tissue. This effect is mediated through the ERβ pathway, as indicated by the absence of LCN2 upregulation when an ERβ antagonist is present [ 73 ]. E2 has been shown to inhibit ferroptosis in the hippocampus by upregulating dihydroorotate dehydrogenase (DHODH), a protein involved in reducing oxidative stress, thereby mitigating memory decline after OVX. These effects are likely mediated by ERs, which activate signaling pathways that enhance DHODH, reduce lipid peroxidation and protect neurons from degeneration. Together, these findings highlight the neuroprotective role of estrogen in ferroptosis regulation and its therapeutic potential for preventing cognitive decline. Catechol estrogens, including 2-hydroxyestrone, 2-hydroxyestradiol, 4-hydroxyestrone, and 4-hydroxyestradiol, have been shown to protect HT22 neuronal cells from chemically induced ferroptosis. These estrogens inhibit ferroptosis by targeting protein disulfide isomerase (PDI), a key mediator of ferroptosis induced by erastin or RSL3 [ 74 ]. Quercetin, through its estrogenic activity, alleviates mitochondrial damage associated with ferroptosis by increasing the level of proteins such as GPX3 and SLC7A11, contributing to the amelioration of depressive behaviors [ 75 ]. Raloxifene, a selective estrogen receptor modulator (SERM), prevents chemically induced ferroptosis in neuronal cells by inhibiting the protein disulfide isomerase (PDI) pathway. By binding directly to PDI, raloxifene provides neuroprotection through the reduction of lipid peroxidation [ 76 ]. This suggests that estrogen receptors may serve as therapeutic targets for preventing ferroptosis-related neurodegenerative diseases. Numerous studies highlight the importance of estrogen signaling via ERs in the context of Alzheimer’s disease (AD), where estrogen appears to exert neuroprotective functions. These functions decline significantly in postmenopausal women, who exhibit an increased predisposition to developing AD compared to men, partly due to altered hormonal balance and decreased estrogen levels [ 77 ]. It has been reported that brain tissues from both AD mouse models and AD patients display features associated with ferroptosis, including abnormal iron metabolism, glutamate excitotoxicity, and lipid ROS accumulation. Elevated iron levels have been observed in the hippocampus, cortex, and basal ganglia of AD patients compared to controls, with brain iron and ferritin levels correlating with amyloid deposition [ 66 ]. Notably, BBB integrity disruption in AD has been shown to occur independently of amyloid-beta (Aβ) and tau pathology, emerging several years before cognitive dysfunction symptoms. This disruption is considered an early biomarker of cognitive decline [ 78 ]. Estrogen signaling may influence BBB integrity, warranting further exploration in the context of AD and other neurodegenerative diseases. Studies on brain metastases have demonstrated that ERβ activation by selective agonists promotes the level of key tight-junction proteins, enhancing BBB integrity and reducing permeability [ 79 ]. Neuroprotective effects of ER agonists have also been demonstrated in animal models using OVX female Wistar rats [ 80 ]. ERα mediates neuroprotection in AD by maintaining intracellular signaling cascades [ 77 ], and reduced ERα levels in hippocampal neurons of AD patients were reported. Similarly, decreased ERβ level in female AD patients has been linked to mitochondrial dysfunction and increased oxidative stress markers [ 81 ]. Emerging scientific evidence increasingly suggests a connection between ferroptosis and AD progression [ 82 ]. Investigating the role of ERs in ferroptosis may offer valuable insights for predicting and potentially treating AD [ 66 ]. Exogenus activations of estrogen receptors may offer a promising therapeutic strategy for AD in women. A deeper understanding of estrogen signaling, ER genes expression, and estrogen metabolism could help identify novel biomarkers and clarify the mechanisms underlying estrogen-mediated neuroprotection. ERα and GPER1 play a crucial role in neuroprotection and regulation of autophagy in neurons. Selective antagonists have been shown to enhance triclocarban’s neurotoxic effects by inhibiting ER-mediated neuroprotection. Triclocarban reduced ERα and GPER1 expression, disrupting estrogen-dependent signaling pathways. This reduction of expression of genes encoding ERα and GPER1 diminishes the neuroprotective capacity of these receptors, amplifying triclocarban’s neurotoxic effects [ 83 ]. Activation of GPER1 improves neurological outcomes and reduces infarct size following cerebral ischemia in mice. This activation increases Nrf2 and GPX4 levels, which protect against oxidative stress and ferroptosis. Inhibition of Nrf2 attenuates the neuroprotective effects, suggesting that GPER1 protects neurons primarily by upregulating Nrf2 and GPX4. This effect also reduces iron accumulation and malondialdehyde formation in stroke models and PC12 cells exposed to oxygen-glucose deprivation and reoxygenation [ 84 ]. Additional evidence linking Nrf and ER has been identified in studies showing that Nrf2 knockout triggers a sex-dependent functional colon phenotype resembling inflammatory bowel disease (IBD). Considering the growing understanding of the gut-brain axis and the influence of gut microbiome dysbiosis on neurodegenerative disease susceptibility, these findings are particularly relevant. NRF2 transcriptional activity has been shown to regulate 17β-estradiol levels as well as ER expression and localization. Administration of 17β-estradiol to female mice via subcutaneous implants normalized colonic motility and alleviated disease symptoms. These findings suggest that targeting estrogen signaling may represent a promising therapeutic strategy for mitigating colonic dysfunction [ 85 ]. Furthermore, other studies have explored the role of ERβ in suppressing colitis development and, consequently, reducing anxiety-like behaviors through the gut microbiome remodeling [ 86 ]. Early studies also highlighted the protective role of GPER1 activation in neurons of the paraventricular nucleus of the hypothalamus and its anti-inflammatory effects in the colon [ 87 ]. Although specific studies in this area remain relatively scarce, the influence of estrogen and its receptors on the gut-brain axis is becoming increasingly evident [ 86 ]. Finally, the effects of sex should be underlined. As indicated above, estrogen signaling pathways play important roles in neuroprotection, as well as in the control of autophagy and ferroptosis. In the light of differences between levels of estrogens and abundance of ERs in males and females, these processes could reveal various specificities of regulation. In turn, such differences might contribute to sex-related differences in frequency and the course of neurological diseases. Indeed, such a phenomenon is observed in the case of many such disorders, which can be exemplified by stroke [ 88 , 90 , 92 ] and Alzheimer disease [ 91 , 92 ]. Under physiological conditions, estrogen regulates various cellular processes, including autophagy, proliferation, apoptosis, survival, differentiation, and vasodilation. It also influences Ca²⁺ mobilization, PI3K signaling, and the MAPK pathway through membrane-bound ERs, exerting nongenomic effects. These effects are particularly evident in endometrial cancer, where activation of the ERα receptor and its interaction with GPER1 promote the proliferation of cancer cells through the PI3K and MAPK signaling pathways [ 51 ]. Erα, in conjunction with NEDD4L, modulates the expression of SLC7A11 . This modulation influences ferroptosis in breast cancer cells post-radiation treatment, suggesting a pathway through which ERα can induce ferroptosis [ 93 ]. GPER1 activation in non-small cell lung cancer cells triggers the PI3K/AKT/mTOR signaling pathway, leading to increased level of stearoyl-CoA desaturase-1. This upregulation prevents ferroptosis, highlighting GPER1’s role in cell survival mechanisms [ 53 ]. Studies show that in estrogen receptor-positive (ER+ - expressing ERα but not ERβ),) breast cancer, estrogen regulates the expression of genes encoding the enzyme MBOAT1, which functions as an inhibitor of ferroptosis by incorporating MUFA into phospholipids. This modification reduces the susceptibility of phospholipids to oxidative damage, thereby enhancing cellular resistance to ferroptosis. In this context, cancer cells exhibit strengthened protective mechanisms, resulting in increased resistance to anticancer therapies. Inducing ferroptosis or employing estrogen antagonists could potentially yield desirable therapeutic outcomes in such cases [ 57 ]. Genistein, through binding to ERα, exerts protective and anti-inflammatory effects in Sjögren’s syndrome (SS). This action is associated with inhibiting ferroptosis mediated by the Xist/ACSL4 pathway. Estrogen receptor activation helps preventing ferroptosis, contributing to the survival of salivary gland cells and reducing inflammation [ 94 ]. Apigenin, another phytoestrogen, activates ERα signaling to alleviate ferroptosis in salivary gland epithelial cells in SS. The mechanism involves regulation of the ATF3/SLC7A1 axis, which is critical for cell survival. This protective effect of apigenin may offer a therapeutic approach for managing xerostomia associated with SS [ 95 ]. ERβ plays a crucial role in regulating ferroptosis - ERβ regulates ferroptosis in intestinal epithelial cells, providing a protective role in inflammatory bowel disease. Activation of ERβ inhibits ferroptosis, reducing inflammation and tissue damage, particularly in the context of colitis. This effect highlights the potential therapeutic role of ERβ activation in regulating ferroptosis and promoting intestinal health. ERβ deficiency exacerbates colitis and enhances ferroptosis, while its activation upregulates GPX4 level, thereby mitigating ferroptosis and reducing inflammation [ 96 ]. ERβ has been implicated in the modulation of ferroptosis-related pathways. Emerging evidence suggests its involvement in regulating oxidative stress and iron metabolism, though further research is needed to clarify these mechanisms [ 57 ]. ER activation inhibits sulfasalazine-induced ferroptosis in breast cancer cells. This occurs via the ERs inhibitory effect on the transferrin receptor, a key protein involved in iron uptake. By modulating iron metabolism, estrogen reduces ferroptosis, potentially offering protection to cancer cells against the damaging effects of ferroptosis [ 97 ]. DN200434 , an orally available inverse agonist of estrogen-related receptor γ (ERRγ), induces ferroptosis in sorafenib-resistant hepatocellular carcinoma (HCC) cells. Research suggests that targeting ERRγ with DN200434 could overcome resistance to sorafenib by triggering ferroptosis, highlighting the potential role of estrogen-related receptors in regulating ferroptosis in cancer cells [ 98 ]. In the MDA-MB-231 and T47D cell lines, ferroptosis was induced using sulfasalazine (SAS), demonstrating that ERs modulate the sensitivity of cancer cells to ferroptosis by influencing TFRC expression and ROS levels. The expression of the TFRC gene is impaired in ER+ tissues, leading to reduced iron accumulation and lower ROS production. As a result, ER+ cells are less sensitive to ferroptosis and exhibit greater resistance to cancer therapies. In contrast, ER− cells display higher efficiency of TFRC production, driving increased ROS production and leading to cell death. Therefore, ER levels may serve as a critical biomarker for predicting the efficacy of this therapeutic strategy [ 97 ]. In similar studies conducted on four breast cancer cell lines and the non-malignant counterpart MCF10A, it was also confirmed that high levels of ERα correlate with reduced sensitivity of breast cancer cells to ferroptosis induced by ionizing radiation (IR), a sensitivity that was restored upon ERα downregulation. ERα was shown to regulate CD71 levels, a key transferrin receptor involved in iron transport into cells, through a mechanism dependent on the E3 ubiquitin ligase NEDD4L. ERα enhances the binding of NEDD4L to CD71, promoting its degradation, thereby reducing intracellular iron accumulation and suppressing ferroptosis, ultimately impairing the effectiveness of therapy. Consequently, the ERα/NEDD4L/CD71 axis has been proposed as a potential therapeutic target in breast cancer radiotherapy [ 99 ]. In another study, the importance of Erα and NEDD4L in ferroptosis was corroborated by demonstration that NEDD4L interacts with a ferroptosis-related protein SLC7A11, and this interaction can be enhanced under conditions of IR [ 93 ]. Subsequently, SLC7A11 is ubiquitinated and efficiently degraded, which indicates another aspect of the influence of the above mentioned factors in ferroptosis regulation. In the context of cancers such as castration-resistant prostate cancer (CRPC), activation of the ERα-NRF2 axis may contribute to therapy resistance, including resistance to bicalutamide. This occurs through enhanced cellular capacity to cope with oxidative stress and the inhibition of ferroptosis, ultimately leading to treatment failure. Therefore, modulation of this signaling axis represents a potential therapeutic target for overcoming resistance in cancers refractory to conventional treatments, particularly in the context of ferroptosis. In summary, the interplay between ERα and NRF2 in the context of ferroptosis may play a crucial role in regulating protection against oxidative stress and modulating cellular damage responses. This is particularly relevant in the development of therapy resistance and the progression of diseases such as prostate cancer [ 100 ]. Ferroptosis is emerging as a potential mechanism leveraged in anticancer therapies and is under intensive investigation in this context. However, certain cancers demonstrate resistance to ferroptosis, posing a challenge to its therapeutic application. Modulation of estrogen receptor activity may offer a promising strategy to overcome this resistance and enhance the efficacy of ferroptosis-based treatments. Recent studies have highlighted the close link between ferroptosis and autophagy, showing that multiple genes and signaling pathways regulate both processes. This functional overlap suggests that autophagy may modulate ferroptotic sensitivity. In this review, we further explore the potential role of estrogen receptors, aiming to elucidate how hormonal signaling may intersect with ferroptotic and autophagic mechanisms, with implications for diseases such as cancer, neurodegeneration, and metabolic disorders [ 101 ]. Autophagy is a fundamental intracellular degradation and recycling mechanism that enables the removal of damaged proteins and organelles, thereby maintaining metabolic homeostasis. It involves the formation of autophagosomes that enclose cellular components destined for degradation, which subsequently fuse with lysosomes to break down their contents. Although autophagy generally serves a protective, adaptive function, excessive activation or impaired regulation can disrupt cellular integrity and ultimately contribute to cell death [ 102 ]. A specialized form of this process is mitophagy—the selective elimination of dysfunctional mitochondria. Mitophagy reduces oxidative stress, preserves efficient energy production, and maintains overall metabolic stability. Because mitochondria are a major source of reactive oxygen species and play a central role in iron handling and lipid metabolism, mitophagy represents a critical link between autophagy and the modulation of cellular sensitivity to ferroptosis [ 103 ]. Studies using the SK-N-MC neuroblastoma cell lines and Erα-positive MCF-7 breast cancer cell lines have revealed an intriguing ERα-specific mechanism that promotes autophagy independently of classical mTOR, PI3KCIII, and MAPK pathways. An inhibition of these pathways did not suppress autophagic activity. Instead, this process appears to be mediated by alternative regulators such as BAG3, ATG7, and WIPI. These findings suggest that ERs can differentially regulate the transcription of autophagy-related genes in cancer cells. Moreover, ERα-driven autophagy has been shown to enhance cellular resistance to oxidative stress, which in the context of our study, may also contribute to protection against ferroptosis [ 36 ]. In the study of TAD1822-7 in a breast cancer cell model, ERβ was involved in inducing cell death through mitochondrial dysfunction and disruption of late-stage autolysosome formation. TAD1822-7 increased ERβ level, leading to cell death, mitochondrial dysfunction, and autophagy, with elevated levels of LC3-II and p62 suggesting a blockade of autolysosome formation. Activation of the PI3K/AKT signaling pathway was also implicated in these effects. High level of ERβ enhanced these responses, while siRNA-mediated inhibition of ERβ abolished cell death and inhibited autophagy. Additionally, TAD1822-7 modulated HIF functions and autophagy by inhibiting HIF-1β under hypoxic conditions. These findings highlight ERβ’s crucial role in regulating autophagy, mitochondrial function, and stress responses, suggesting that its activation can lead to cellular imbalance and cell death [ 104 ]. ERα has been demonstrated to regulate mitochondrial biogenesis and mitophagy, which are essential processes for maintaining cellular health. Genistein, through its activation of ERα, stimulates these processes, counteracting mitochondrial dysfunction observed in BMMSCs exposed to oxidative stress. This highlights not only the antioxidative role of ERα but also its involvement in autophagy, particularly mitophagy, influencing mitochondrial integrity and functionality. The mechanism underlying this action involves genistein-induced activation of ERα, which increases the levels of key antioxidative regulators such as sirtuin 3 (SIRT3) and PGC1α [ 105 ]. ERs, particularly ERβ, also play a key role in regulating anti-inflammatory responses and repair mechanisms in colitis by activating NLRP6 and promoting autophagy. ERβ physically interacts with the nucleotide-binding domain of NLRP6, facilitating the assembly of the NLRP6 inflammasome. The ERβ-NLRP6 axis interacts with key autophagy-related proteins (ULK1, BECN1, LC3B, ATG16L1, p62), regulating autophagosome biogenesis and autophagic flux. Furthermore, ERβ indirectly regulates processes related to the polyubiquitination of inflammation-related proteins (ASC, Casp-1 p20, IL-1β, TNF-α) via NLRP6-initiated autophagy. This reduces the levels of inflammatory mediators and facilitates the restoration of tissue homeostasis [ 106 ]. Additionally, in colon cancer cells, ERβ regulates the cell cycle by inhibiting proliferation through the reduction of cyclin D1 levels. The accumulation of cyclin D1 in cells with impaired autophagy disrupts homeostasis. ERβ activates autophagy, including the mTOR pathway and BNIP3 protein, promoting the degradation of cyclin D1 and controlling cell proliferation, thereby limiting tumor growth [ 107 ]. Recent studies suggested that starvation-induced autophagy varies by sex, with a stronger response in females, underscoring the importance of considering sex in research and therapy development. ERs, especially ERα, regulate autophagy-related genes in this process. In ERα and ERβ knockout medaka fish, autophagy processes were altered depending on the presence of the receptor. ERs also regulate pathways such as mTOR and HK2. In ERα-KO fish, starvation increased mTOR transcription in males and reduced HK2 and ULK activity in both sexes. Furthermore, ERs affect the nuclear-cytoplasmic translocation of LC3, with disruptions observed in the knockout fish. These results suggest that ER availability and sex influence autophagosome formation and autophagic flux [ 108 ]. In in vitro studies using human umbilical vein endothelial cells (HUVEC) and in vivo models have shown that E2 activates ER-α, leading to an increase in autophagosome formation, as indicated by elevated LC3 levels. It also reduces lysosomal fusion with autophagic vesicles, as indicated by low p62 level. Activation of ER may provide protection against atherosclerosis and cardiovascular diseases, which are increasingly becoming a significant concern in society [ 109 ]. While the role of ERs in cancer has been previously discussed, we would like to highlight findings where, in ER-negative (MDA-MB-231) cells, gemcitabine induces autophagy with a cytoprotective character. In contrast, in ER+ cells (MCF-7), autophagy is cytotoxic. The ERα-ERK-p62 signaling pathway is involved in this process in MCF-7 cells, where its activation leads to increased autophagic degradation and subsequent cell death. Inhibition of this pathway, for example, using small interfering RNAs, impairs autophagic degradation, switching the autophagic response from cytotoxic to cytoprotective. Furthermore, high level of ERα in ER-negative breast cancer cells (BCap37) induces autophagy and switches the autophagic response from cytoprotection to cytotoxicity in the presence of gemcitabine. These findings again emphasize the regulation of autophagy and the modulation of autophagic mechanisms, depending on ER status, and highlight the involvement of ERK and P62 [ 110 ]. ERα play a key role in the pathogenesis of uterine leiomyomas (ULM), particularly in the HMGA2-ULM subtype. The level of ERα correlates with the levels of HMGA2 protein, which stimulates ULM cell proliferation. Additionally, the interaction of p62 with ERα suggests that the autophagy pathway influences ERα level and ULM cell viability. Inhibition of this pathway through the blockade of the HMGA2-p62-ERα axis may represent a promising therapeutic strategy for treating HMGA2-ULM [ 111 ]. It was reported that ERs play also a crucial role in the cardioprotective effects of E2, particularly in regulating mitochondrial homeostasis, mitophagy, and autophagy in the heart [ 110 ]. In that study, estrogen treatment in OVX or sham-treated Fulvestrant (ER antagonist) rats restored lipid metabolism, AMPK signaling, and mitochondrial dynamics, all of which were disrupted in the absence of estrogen. Application of Fulvestrant blocked most of these beneficial effects, indicating that ER-mediated signaling is essential for restoring autophagic and mitophagic processes, both of which are closely linked to ferroptosis [ 112 ]. These findings highlight the significance of ER signaling in cardioprotection, particularly in the postmenopausal state, and suggest that receptor-independent mechanisms may also play a role in regulating ferroptosis, which warrants further investigation. Estrogen deficiency leads to a reduction in ERα levels, affecting various cellular processes, such as the activation of the AKT-mTOR pathway and autophagy mechanisms, impacting neuronal health. In OVX rats, excessive autophagy is observed in the hippocampus, characterized by elevated levels of autophagic proteins and the presence of autophagosomes. Simultaneously, there is inhibition of AKT-mTOR signaling, resulting in decreased levels of HB-EGF and p-EGFR in the hippocampus. This may contribute to neuronal apoptosis and cognitive decline. The administration of E2 and HB-EGF in OVX rats restored EGFR activation in the hippocampus, reducing excessive autophagy and preventing neuronal loss [ 113 ]. As increasing evidence highlights the interplay between mitophagy, autophagy, and ferroptosis [ 114 , 116 ], with the examples provided above. These processes appear to be interconnected indirectly through shared regulatory genes and proteins. Table  1 summarizes selected key molecules most frequently implicated in the crosstalk between ferroptosis, autophagy, and mitophagy. Table 1 Common genes/proteins for ferroptosis, autophagy and mitophagy Gene and protein Ferroptosis Autophagy Mitophagy SQSTM1 p62 Involvement in ferroptosis regulation through the KEAP1-NRF2 pathway, ROS generation, and redox balance Regulation of autophagosome biogenesis and autophagic flux Recognition of ubiquitinated mitochondria and their targeting to autophagosomes BNIP3 BNIP3 Promotes the degradation of cyclin D1, preventing the cell from passing through the G1/S checkpoint, thereby inhibiting cell proliferation. It activates the p62-Keap1-NRF2 pathway, regulating redox balance Associated with autophagy via BCL-2 proteins, and linked to the mTOR pathway Key for the initiation of mitophagy, helps in the removal of damaged mitochondria regulating redox balance MAP1LC3 LC3 Participates in the formation of autophagosomes, mediates ferritin degradation, and iron release Autophagy marker, a key protein involved in the formation and development of the autophagosome Essential for the formation of autophagosomes that surround damaged or unnecessary mitochondria NFE2L2 NRF2 Interacts with pathways such as Ras/RAF/MEK/ERK and Ras/PI3K/PTEN/Akt. Akt inhibits GSK3β, stabilizing NRF2 and enhancing its nuclear import and antioxidant activity. At the same time, KEAP1 must be blocked or degraded. NOX1 inhibits NRF2 activity and leads to ferroptosis Oxidative stress response and autophagy regulation, it controls the expression of antioxidant genes such as HO-1, NQO1, GSTs, TrxR1, and GPX4 Oxidative stress response, essential for the formation of autophagosomes, regulates mitochondrial quality, interacts with proteins such as PINK1 and Parkin, where PINK1 accumulates on damaged mitochondria PRKN Parkin Associated with the removal of damaged mitochondria, may influence the regulation of ferroptosis Helps in removing damaged organelles in cells and supports autophagy. Initiates the degradation of damaged or unnecessary cellular components through ubiquitination of proteins, which are directed to autophagosomes and degraded by lysosomes Works via PINK1/Parkin pathway, marking damaged mitochondria for selective degradation. PINK1/Parkin recruits autophagosomal membranes through Rab and LC3 proteins, followed by lysosomal degradation. PINK1 activates Parkin by phosphorylation GPX4 GPX4 Plays a crucial role in preventing ferroptosis by reducing lipid peroxides in cellular and mitochondrial membranes, using glutathione as a cofactor to inhibit lipid peroxidation and oxidative damage Maintains redox balance and protects the cell from oxidative stress Maintains mitochondrial health by removing excess peroxides, playing a protective role in mitophagy through the stabilization of mitochondrial membranes and preventing lipid peroxidation in mitochondria Common genes/proteins for ferroptosis, autophagy and mitophagy SQSTM1 p62 BNIP3 BNIP3 NFE2L2 NRF2 PRKN Parkin GPX4 GPX4

Ferroptosis is a regulated, iron-dependent form of cell death characterized by uncontrolled peroxidation of membrane phospholipids. This process plays an essential role in maintaining tissue homeostasis by eliminating cells with impaired lipid metabolism or disrupted redox balance. Unlike apoptosis or necroptosis, ferroptosis does not involve caspase activation or the inflammatory processes involving typical inflammatory mediators. The mechanism of ferroptosis is driven by reactive oxygen species (ROS) generated via the Fenton reaction, where ferrous ions (Fe²⁺) catalyze the oxidation of polyunsaturated fatty acids (PUFAs). A critical defense against ferroptosis is provided by glutathione peroxidase 4 (GPX4), an enzyme that neutralizes lipid peroxides, preventing their toxic accumulation [ 1 ]. Under physiological conditions, ferroptosis prevents the accumulation of dysfunctional cells, particularly in organs with high iron turnover and lipid metabolic activity. Growing evidence indicates, however, that dysregulation of ferroptosis contributes to the pathogenesis of numerous diseases. Excessive activation of this pathway has been observed in neurodegenerative disorders (e.g., Parkinson’s disease, Alzheimer’s disease), acute kidney injury, ischemic stroke, and liver diseases. Conversely, suppression of ferroptosis promotes survival of cancer cells and contributes to therapeutic resistance, particularly in tumors with high metabolic activity and increased iron utilization [ 2 ]. Emerging evidence indicates that ferroptosis is closely intertwined with autophagy, a cellular degradation and recycling process that maintains homeostasis by removing damaged organelles and proteins. Selective forms of autophagy, such as mitophagy and ferritinophagy, can modulate ferroptotic sensitivity by regulating mitochondrial function, ROS levels, and intracellular iron availability. These findings suggest that the balance between ferroptosis and autophagy is a critical determinant of cell fate under stress conditions, linking metabolic, oxidative, and hormonal signaling pathways [ 3 ]. Estrogen/ER signaling regulates cellular metabolism, redox balance, and survival pathways through receptors ERα, ERβ, and GPER. Evidence can be find in the literature that estrogen can modulate both autophagy and ferroptosis by affecting lipid peroxidation, antioxidant defense, iron metabolism, and the regulation of autophagy-related proteins. In estrogen-dependent conditions such as endometriosis or endometriotic cysts, estrogen influences autophagy and ferroptosis, indicating a crosstalk shaped by the hormonal environment [ 4 – 6 ]. Similar regulatory effects on both autophagy and ferroptosis have been observed in endometrial cancer, and postmenopausal atherosclerosis [ 7 , 8 ]. These findings underscore how estrogen/ER signaling modulates the crosstalk between ferroptosis and autophagy, highlighting its implications for physiology and disease.