*Review* **Ferroptosis and Its Potential Role in Glioma: From Molecular Mechanisms to Therapeutic Opportunities**

**Yusong Luo 1,2,3, Guopeng Tian 1,2,3, Xiang Fang 1,2,3, Shengwei Bai 1,2,3, Guoqiang Yuan 1,2,3,\* and Yawen Pan 1,2,3,\***


**Abstract:** Glioma is the most common intracranial malignant tumor, and the current main standard treatment option is a combination of tumor surgical resection, chemotherapy and radiotherapy. Due to the terribly poor five-year survival rate of patients with gliomas and the high recurrence rate of gliomas, some new and efficient therapeutic strategies are expected. Recently, ferroptosis, as a new form of cell death, has played a significant role in the treatment of gliomas. Specifically, studies have revealed key processes of ferroptosis, including iron overload in cells, occurrence of lipid peroxidation, inactivation of cysteine/glutathione antiporter system Xc− (xCT) and glutathione peroxidase 4 (GPX4). In the present review, we summarized the molecular mechanisms of ferroptosis and introduced the application and challenges of ferroptosis in the development and treatment of gliomas. Moreover, we highlighted the therapeutic opportunities of manipulating ferroptosis to improve glioma treatments, which may improve the clinical outcome.

**Keywords:** glioma; ferroptosis; lipid peroxidation; molecular mechanisms; treatment

#### **1. Introduction**

Glioma is the most common primary malignant tumor of the brain, accounting for approximately 50–60% of the central nervous system (CNS) tumors [1] and approximately 81% of intracranial malignancies [2,3]. Patients with gliomas have significantly higher recurrence rates than those with other tumors of the CNS [4]. Gliomas have been classified by the World Health Organization (WHO) grading system into four grades, where gliomas of grade 1 and grade 2 indicate low-grade gliomas, and gliomas of grade 3 and grade 4 reveal high-grade gliomas [5]. The median overall survival (OS) time of low-grade glioma patients is approximately 11.6 years [6]. However, patients with grade 3 glioma have a median OS time of approximately three years, and the median OS time of grade 4 glioma patients is approximately 15 months [7]. While current clinical treatments for glioma consist of surgical resection, radiotherapy, chemotherapy, novel molecular targeted therapy and immunotherapy [8], these treatments have not brought desirable benefits to patients, and the prognosis of patients remains extremely poor [9,10]. Therefore, there is a great need to develop new therapeutic strategies, including novel therapeutic targets inhibiting glioma cells, to improve OS time and the quality of life for these patients. The common deaths of different cells in the body include necrosis, apoptosis, autophagy and pyroptosis [11]. Recently, ferroptosis, as a new nonapoptotic cell death pattern resulting from iron-dependent lipid peroxidation injury, has attracted more attention [12–14].

Ferroptosis is a new type of programmed cell death triggered by cell membrane damage arising from these processes, including intracellular iron accumulation, production of reactive oxygen species (ROS), lipid peroxidation, failure activity of glutathione peroxidase (GPX) and xCT [15,16]. Cells undergoing ferroptosis not only have changes in cell composition, but also in cell morphology. When cells undergo ferroptosis, although the

**Citation:** Luo, Y.; Tian, G.; Fang, X.; Bai, S.; Yuan, G.; Pan, Y. Ferroptosis and Its Potential Role in Glioma: From Molecular Mechanisms to Therapeutic Opportunities. *Antioxidants* **2022**, *11*, 2123. https://doi.org/10.3390/ antiox11112123

Academic Editors: Yan-Zhong Chang and Stanley Omaye

Received: 13 September 2022 Accepted: 26 October 2022 Published: 28 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

morphology of the nucleus does not change significantly, the morphology of mitochondria shows increased bilayer membrane density, reduction or disappearance of cristae, and reduced volume [16,17]. Many recent studies have shown that significant progress has been made on the impacts of ferroptosis on glioma. Ferroptosis inducers, as compounds from plants and others, have certain effects on the treatment of glioma by affecting ferroptosis processes.

Herein, we will mainly focused on the essential molecular mechanisms of ferroptosis, as well as the potential impact of ferroptosis on glioma growth and treatment. We also provided an overview of the challenges of ferroptosis in glioma therapy and discussed the therapeutic opportunities of manipulating ferroptosis to improve treatment.

#### **2. Molecular Mechanisms of Ferroptosis**

J.M. Gutteridge found in 1984 that iron salts could induce lipid peroxidation by breaking down lipid peroxides into alkoxyl and peroxyl radicals, and iron complexed with ethylene diamine tetraacetic acid (EDTA) could also initiate lipid peroxidation by reacting with hydrogen peroxide (H2O2) to form hydroxyl radicals (•OH), which may lay the foundation for iron-dependent cell death [18]. In addition, it has been reported that exogenous glutamate could induce cell death by inhibiting cystine uptake through xCT to lead to decreased glutathione production, and a unique programmed cell death pathway called oxytosis, which was dependent on oxidative stress and ROS production and was introduced [19]. This laid the prior groundwork for the discovery and proposal of ferroptosis. Ferroptosis was defined as a new form of programmed cell death by Brent R. Stockwell in 2012 that expresses the process of iron-dependent cell death in cancer cells [13]. Molecular mechanisms of ferroptosis differ from other major forms of regulated cell death (RCD) (Table 1). The main biochemical processes of ferroptosis consist of excess iron and accumulation of ROS in cells, lipid peroxidation, inactivation of xCT and depletion of glutathione and lipid repair enzyme [20–22].


**Table 1.** The features of different forms of RCD.

#### *2.1. Iron Metabolism*

Iron is a basic trace element for various cells to carry out various biological functions. Dietary iron comes in many forms but is typically classified as either non-heme or heme iron (Fe2+ complexed with protoporphyrin IX). Non-heme dietary iron exists largely as ferric salts, which are reduced back to Fe2+ by iron reductase in the intestine. Fe2+ enters intestinal epithelial cells (IECs) via the brush-border transporter divalent metal transporter 1 (DMT1) and exits through ferroportin 1 (FPN1) in the basolateral membranes [23,24]. Fe2+ is oxidized to Fe3+ by ceruloplasmin (CP) and hephaestin (HP), and Fe3+ combines with transferrin (Tf) to be transported in the blood [25]. Tf-Fe3+ attaches to the transferrin receptor (TfR) on the cell membrane and then internalizes to the cell as endosomes [26,27]. Fe3+ is released and reduced to Fe2+ by 6-transmembrane epithelial antigen of the prostate 3 (STEAP3) in a variety of cells; then, Fe2+ enters the cytoplasm via DMT1 on the endosomal membrane [28,29]. Heme iron is a part of the hemoproteins hemoglobin and myoglobin. However, the molecular mechanism of heme iron absorption is still unclear. There is some evidence that ingested heme iron is decomposed by heme oxygenase in intestinal cells, thus releasing free ferric iron. A large amount of Fe2+ accumulates in the cytoplasm to form a labile iron pool, and the metabolic activity of Fe2+ has a vital impact on various biological functions, such as ferroptosis [30,31]. Intracellular iron overload, with H2O2, triggers the Fenton reaction, inducing the formation of ROS, such as •OH, which cause lipid peroxidation to provoke ferroptosis [32–34] (Figure 1). Iron responsive element binding protein 2 (IREB2), as a significant regulator of iron metabolism, may develop sensitivity to ferroptosis [35–37]. Meanwhile, autophagy can regulate the iron pool by affecting the recruitment of ferritin to autophagosomes for lysosomal degradation to release free iron [38–40]. For example, ferritinophagy directly recognizes the ferritin heavy chain 1 (FTH1) by the cargo receptor nuclear receptor coactivator 4 (NCOA4) and then releases iron by transporting the ferritin complex to autophagosomes for lysosomal degradation [41]. Conversely, reduced intracellular Fe2+ levels could impede the process of ferroptosis. For instance, erastin-induced ferroptosis was weakened by decreased intracellular Fe2+ because of the knockout of autophagy-related 5 (ATG5) or autophagy-related 7 (ATG7) [42]. Thus, the metabolism of iron plays a vital role in ferroptosis.

**Figure 1.** Iron absorption and metabolism in the body. Fe2+, ferrous cation; Fe3+, ferric cation; Dcytb, duodenal cytochrome b; DMT1, divalent metal transporter 1; FPN1, ferroportin 1.

#### *2.2. Lipid Metabolism*

Lipid peroxidation, a hallmark feature of ferroptosis, is the ultimate executor of ferroptosis. ROS generated by the Fenton reaction interact with polyunsaturated fatty acids (PUFAs) on cellular or organelle membranes to generate toxic phospholipid hydroperoxides (PLOOHs), thereby inducing ferroptosis [43–45]. Research has shown that some factors, such as acyl–coenzyme A synthetase long-chain family 4 (ACSL4), lysophosphatidylcholine acyltransferase 3 (LPCAT3) and lipoxygenases (LOXs), participate in the production of lipid

peroxidation [46–48]. ACSL4 (as a required lipid metabolism enzyme) and LPCAT3 (as a class of key enzymes catalyzing the reacylation of lysophospholipids to phospholipids) activate free long-chain polyunsaturated fatty acids, promote lysophosphatidylcholine (LPC) conversion into lecithin, mediate the synthesis of oxidized cell membrane phospholipids, and subsequently regulate ferroptosis development [49,50]. Meanwhile, ACSL4 esterifies arachidonic acid (AA) into acyl-coenzyme A (acyl-CoA) for the biosynthesis of PUFAs, which plays a key role in lipid peroxidation and ferroptosis [51]. LOXs, pivotal regulators of ferroptosis, may have a vital effect on the initiation of ferroptosis by promoting lipid autoxidation and predicting ferroptosis sensitivity [47,52]. Therefore, lipid peroxidation in ferroptosis executes cell death by the destruction of the lipid bilayer on cellular or organelle membranes (Figure 2a).

**Figure 2.** Molecular mechanisms of ferroptosis. (**a**) The lipid metabolism pathway; (**b**) the xCT/GPX4 pathway. ACSL4, long-chain fatty acid CoA ligase 4; PUFA, polyunsaturated fatty acid; LPCAT3, lysophosphatidylcholine acyltransferase 3; PUFA-CoA, polyunsaturated fatty acid coenzyme A; ALOXs, lipoxygenases; PUFA-PL, polyunsaturated fatty acid-containing phospholipid; TCA, tricarboxylic acid cycle; GSH, glutathione; GSSG, glutathione disulfide; GPX4, glutathione peroxidase 4.

#### *2.3. The xCT and GPX4*

Environmental pressure (such as high temperature and hypoxia) can cause iron reaction, so the cell also needs to establish an appropriate mechanism of defense ferroptosis. The most classic defense way of ferroptosis is the antioxidant axis formed by xCT, glutathione (GSH), and GPX4. The xCT, as a transmembrane protein, consists of light-chain solute carrier family 7 member 11 (SLC7A11) and heavy-chain solute carrier family 3 member 2 (SLC3A2, CD98hc or 4F2hc). SLC7A11, which is a main functional subunit of xCT, aims to regulate extracellular cysteine (Cys) into cells and intracellular glutamic acid (Glu) out of cells, and SLC3A2, as an important subunit, maintains the stability of xCT by anchoring and stabilizing SLC7A11 [53,54]. Then, Cys generates reduced GSH with Glu and glycine (Gly) under the catalysis of glutamate cysteine ligase (GCL) and glutamylcysteine synthetase (GCS) [55,56]. Beclin 1 suppresses xCT activity to promote ferroptosis by adhering to SLC7A11 directly [57]. GPX4, as a key enzyme in ferroptosis, reduces PLOOH to nontoxic phospholipid alcohols (PLOHs) in membranes with GSH to prevent ferroptosis [58]. The inhibitor 2-cyano-3,12-dioxooleana-1,9 (11)-dien-28-oic acid (CDDO) prevents the specific degradation of GPX4 via chaperone-mediated autophagy (CMA) by affecting the interaction between heat shock protein 90 (HSP90) and lysosomes, which inhibits the ferroptosis of cells [59]. However, the inhibition of CMA is relieved by inhibiting the mammalian target of rapamycin (MTOR) pathway [60], which may involve the degradation of GPX4 to promote ferroptosis. Therefore, xCT, GPX4 and GSH could be regulated and have an important effect on ferroptosis (Figure 2b).

#### *2.4. FSP1 and DHODH*

In addition to the classic GPX4 defense pathway, recent studies have identified ferroptosis suppressor protein 1 (FSP1) and dihydroorotate dehydrogenase (DHODH) independent of the GPX4 signaling pathway. They all involve ubiquinone, a metabolite molecule that exists in both chemically reduced and oxidized states. Ubiquinone (or CoQ) is a lipid that functions on cell membranes and mitochondrial membranes. FSP1 in the cell membrane inhibits ferroptosis by reducing ubiquitin to ubiquinol (CoQH2), which acts as a free radical trapping antioxidant to prevent lipid peroxidation at the cell membrane [61,62]. Similar to the FSP1 system mechanism, DHODH-mediated regulation of panthenol production is an effective system that is specifically designed to alleviate lipid peroxidation in the mitochondria. Mitochondria produce a large amount of ROS in the electron transport chain located in the inner membrane during oxidative phosphorylation. Lipid peroxidation occurs when the mitochondrial antioxidant system is damaged and unable to remove ROS. DHODH is a flavin-dependent enzyme located in the mitochondrial inner membrane and its main function is to catalyze the fourth step of the pyrimidine biosynthesis pathway [63]. The oxidation of dihydroorotate (DHO) to orotate (OA) simultaneously transfers electrons to the ubiquitin in the inner mitochondrial membrane for its reduction to ubiquinol. Gan's team found that when cells were treated with GPX4 inhibitors, such as RSL3, metabolomic analysis revealed a significant decrease in N-aminoformyl-aspartate (C-Asp), increase in Uridine and increase in the synthesis of uridine 5- -monophosphate (UMP) [64]. This suggests a possible relationship between ferroptosis and pyrimidine nucleotide synthesis. By supplementing intermediate metabolites for pyrimidine synthesis, the authors found that dihydroorotic acid inhibited RSL3-induced ferroptosis, whereas orotic acid made cells more sensitive to RSL3. Since DHO and OA are substrates and products of DHODH, respectively, this further confirms that DHODH may be involved in the regulation of ferroptosis. Interestingly, further studies have revealed the use of the DHODH inhibitor Brequinar (BQR) to induce ferroptosis in GPX4 low-expression cells, while the high expression of GPX4, BQR treatment significantly increased the sensitivity of cells to ferroptosis inducers. As is known, most GBM cell lines have higher expression levels of DHODH and GPX4 compared to normal human astrocyte cytoplasm (NHA) [65]. The research also confirms that, in the solid tumor with a high expression of GPX4, the combination of ferroptosis inducer sulfasalazine and DHODH inhibitor can have good therapeutic effect. This finding provides a new strategy for how to target ferroptosis in glioma therapy.

In general, there are at least three types of iron-death defense systems in cells based on different subcellular localization: GPX4 in the cytosol and mitochondria, FSP1 in the cell membrane, and DHODH in mitochondria.

#### **3. Targeting Ferroptosis to Treat Glioma**

#### *3.1. Metabolic Pathway*

Iron in the brain plays a crucial role in maintaining proper functioning of the central nervous system through its participation in many cellular activities, such as myelination, neurotransmitter synthesis, and energy production. The maintenance of this homeostasis depends on the function of the blood–brain barrier (BBB), which is composed of brain microvascular endothelial cells (BMVEC), astrocytes, microglia and pericytes. It has long been thought that the development of the BBB leads to a reduction in iron absorption in

infancy. However, some evidence has revealed that iron levels in the brain increase with ageing [66,67]. Additionally, the mechanism of iron uptake into BMVEC is thought to be primarily via the Tf/TfR1 pathway. Iron was first uptake into BMVEC from blood by TfR1 mediated endocytosis. Then, the action of an H+-ATPase, the membrane of endocytosomes reduced the pH of the endocytosomes, resulting in the dissociation of Fe3+ from Tf and their reduction to Fe2+, which will cross the endosomal membrane by a process mediated by DMT1. When the PH rises to 7.4, the non-iron-bound Tf (apo-Tf) and TfR1 return to the luminal membrane where apo-Tf-TfR1 is released into the blood for the next round of iron uptake [68]. Interestingly, after iron was released into the brain interstitial fluid, it was unevenly distributed among different cell types in different regions of the brain [69]. Almost all iron transport-related proteins are expressed in glial cells, but not in neurons.

As is known, iron is a cofactor for many enzymes, including ribonucleic acid reductase (RR), which is an enzyme involved in DNA synthesis. To maintain proliferation, GBM cells need to increase iron uptake, thereby regulating the expression of proteins involved in iron uptake. Recently, studies have reported that there are higher free iron levels in glioma than in other brain tumors, such as meningioma cells and glioblastoma cancer stem cells [70]. Iron-related gene expression in gliomas, such as TfR1 and TfR2, is different from that in other brain tumors and normal human brain tissue [71,72]. TfR levels in glioma sample tissues appear higher than those in meningiomas and other brain tumors in general, which may be correlated with the high levels of iron in gliomas [73]. In addition, the high expression of TfR2 not only promotes glioma cell proliferation, but also contributes to the better sensitivity to temozolomide. The proliferation of glioma cells is attributed to TfR2, which could be localized in lipid rafts and stimulate the ERK1/ERK2 phosphorylation by combining with Tf, but the mechanism of TfR2-induced glioma hypersensitivity to temozolomide remains unclear [74]. Therefore, the effect of TfR2 on glioma is still controversial, and more reports are needed to verify this. Several recent studies have indicated that iron homeostasis and ferroptosis are also affected by iron-sulfur cluster (ISC) proteins. Loss of ISC could induce ferroptosis by initiating iron-starvation responses to lead to iron overload in tumor cells. This mechanism is that ISC synthesis inhibition could activate the iron regulatory protein (IRP); IRP increases TfR levels and reduces FPN1 production by binding to target mRNAs, which promotes intracellular excessive iron accumulation [75]. However, whether ISC has a similar role in glioma requires more evidence.

DMT1 may be related to increased iron levels in glioma cells and is currently a molecular marker in neurodegenerative diseases [76]. In an experimental rat model with C6 glioma cells, propofol inhibits DMT1 expression, tumor cell proliferation and eventually decreased glioma weight [77]. Additionally, this tumor suppressive effect was further found to be associated with a significant reduction in the GSH and ROS. However, a study showed that temozolomide (TMZ) may suppress tumor growth by inducing ferroptosis by targeting DMT1 expression in glioblastoma cells [78]. These results suggest that DMT1 may affect glioma proliferation by regulating ferroptosis and ROS levels and has been investigated as a potential therapeutic target. While the STEAP3 protein plays a vital role in other processes, such as affecting the inflammatory response by regulating the Toll-like receptor 4-mediated macrophage production of chemoattractant protein-5, interferon-beta and interferon-induced protein-10 [79,80], it could also have an essential impact on ferroptosis by reducing Fe3+ to Fe2+ [81]. The expression of STEAP3 in glioma cells is higher than that in normal brain tissues, which could be regarded as a potential prognostic marker and reduce the overall survival of patients with glioma [82,83]. In addition, STEAP3 not only regulates ferroptosis by enhancing TfR expression and inducing mesenchymal transition, but also has a direct influence on glioma cell proliferation, invasion, and sphere formation in vitro and on glioma growth in vivo [84]. Poly(C)-binding protein 2 (PCBP2), as a significant factor in iron metabolism and posttranscriptional and translational regulation, possibly affects the process of ferroptosis. While PCBP2 is upregulated in glioma tissues and cell lines, the development and proliferation of glioma are suppressed when it is knocked down or when its inhibitor microRNA-214 is applied [85]. The higher levels of ferritin

detected in the serum of patients with tumors possibly predict that the prognosis of these patients will deteriorate more, which indicates that iron metabolism plays a necessary role in the progression and therapy of tumors. Ferritins in the serum and cerebrospinal fluid of patients with gliomas, which could come from glioma cells, were higher than those of patients without gliomas [86]. Significant evidence has suggested that nuclear factor erythroid-related factor 2 (NRF2) acts as a key regulator of antioxidant responses, which favors cancer cell growth and leads to increased drug resistance in tumor cells [87–89]. NRF2 mainly targets heme oxygenase-1 (HO-1) to reduce the levels of ROS and degrade prooxidants [90]. Research has shown that neuronal precursor cell-expressed developmentally downregulated 4-1 (NEDD4-1) induces resistance to TMZ treatment in gliomas via activating the AKT/NRF2/HO-1 axis [91]. TMZ also induces ferroptosis by inhibiting the NRF2/HO-1 signaling pathway in gliomas [78]. NRF2/HO-1axis appears to play an important role in glioma therapy. In addition, triptolide and brusatol, as NRF2 inhibitors, suppresses potently IDH1-mutated glioma cells by targeting the NRF2-driven glutathione synthesis pathway to induce lipid peroxidation [92,93]. The upregulated cystathionineγ-lyase (CSE) in IDH1-mutant astrocytomas promotes cell survival by maintaining GSH to drive antioxidant defense, and whether it is related to the NRF2 needs further verification [94]. Therefore, ferroptosis in gliomas could be associated with the regulation of NRF2.

#### *3.2. The xCT Pathway*

Cysteine deprivation is an important inducer of ferroptosis and greatly contributes to the ferroptosis in GBM [95,96]. The study conducted by Takeuchi et al., including 40 patients with gliomas, concluded that high levels of xCT could predict a short progressionfree survival and a low overall survival [97]. Specifically, the high levels of xCT possibly promote glioma cells to grow and survive by enhancing mitochondrial biogenesis and adenosine tri-phosphate (ATP) generation, as well as by reducing the accumulation of ROS [98]. These findings suggest that we can inhibit system Xc-induces ferroptosis in glioma. Radiation, chemotherapy (such as TMZ) and immunotherapy could lead to the activation of ferroptosis by downregulating the expression of xCT to induce the death of glioma cells [99–101]. Some widely used clinical drugs have also been applied to the treatment of gliomas by managing xCT and sequentially regulating ferroptosis. Gao et al. found that ibuprofen could enhance ferroptosis by depleting the expression of xCT and GPX4 to inhibit the growth of glioma cells [102]. Another study found that sulfasalazine could stimulate ferroptosis by inhibiting the activity of xCT and sequentially decreasing the formation of GSH strengthened the effect of radiation therapy to increase the overall survival of mice [103]. In addition, tumor suppressor P53 is a frequently mutated gene in various cancers, including glioma. P53 suppresses glioma growth by the induction of ferroptosis [104]. Notably, P53 possibly inhibits the activity of xCT by directly depleting the level of SLC7A11, thus promoting the ability of ferroptosis to suppress the growth of glioma cells [105]. However, an interesting phenomenon is that, in glucose deprivation environments, the treatment of epidermal growth factor will upregulate xCT in glioma cell lines, leading to tumor death [106–109].

In summary, xCT could play a dual role in the development and treatment of gliomas, so further studies are needed to express the practical effect of xCT in gliomas.

#### *3.3. GPX4 Expression*

Recently, a study revealed that when GPX4 is knocked down or reacts with its inhibitors, ferroptosis is activated to induce the death of glioma cells by accumulating lipid peroxides to damage the cell membrane and organelle membrane [110]. Some ferroptosis inducers, such as plumbagin, triggers ferroptosis by inducing GPX4 degradation via the lysosome pathway and inhibiting glioma growth [111]. In addition, it has been demonstrated that certain traditional Chinese herbs induce ferroptosis in glioma. For instance, capsaicin, as a potential anticancer ferroptosis inducer, suppresses the proliferative effects of glioma

cells by increasing ACSL4 levels and decreasing GPX4 levels to induce ferroptosis [112]. Dihydrotanshinone I (DHI), which boosted ferroptosis by decreasing the expression level of GPX4 and increasing that of ACSL4, inhibited the growth and proliferation of glioma cells [113]. The curcumin analog ALZ003 inhibited the development and growth of glioma cells and enhanced their sensitivity to temozolomide treatment by promoting the androgen receptor (AR) ubiquitination and downregulating GPX4 to highlight ferroptosis in vitro, which improved the survival of experimental rodents in vivo [114]. Additionally, the natural compound artesunate (ART), as an antimalarial drug, was previously demonstrated and ART also exhibited an anti-tumor effect and was a specific inducer of ferroptosis in a number of different types of cancer, including glioma. It inhibits the proliferation of glioma cells in vitro and in vivo by promoting GSH depletion and low GPX4 expression to increase ferroptosis [115]. In another study, dihydroartemisinin (DHA), as an inhibitor, could have the prospect of treating glioma because of its role in promoting apoptosis and autophagy and reducing the invasion ability of glioma cells [116,117]. Specifically, DHA could promote the development of ACSL4 and xCT, but significantly downregulated GPX4 levels, initiating the death of glioma cells by maintaining ferroptosis [118]. Beyond the above stated aspects, there are many new nanomaterials involved. It was reported that the biomimetic nanoparticles (PIOC@CM NPs) increased the level of ROS, depleted GSH upon ultrasonic irradiation and attenuated the activity of GPX4 to kill glioma C6 cells by activating ferroptosis [119]. Iron oxide nanoparticles loaded with paclitaxel (IONP@PTX) not only inhibited the migration and invasion of glioma cells by enhancing ions, ROS and lipid peroxidation, but also promoted the autophagy-dependent ferroptosis pathway by decreasing the levels of GPX4 in vitro [120]. Above all, these findings provide some new drug treatment options for glioma and demonstrate that GPX4 degradation promotes ferroptosis in glioma.

#### *3.4. Tumor Immune Microenvironment*

As key regulatory components, immune cells and immune-related molecules have been shown to play pivotal roles in the development and treatment of glioma cells. Accumulating evidence indicates that ferroptosis not only promotes tumor cell death, but also affects the tumor immune microenvironment (TIME) [121,122] (Figure 3). As key regulatory components, immune cells and immune-related molecules have been shown to play pivotal roles in the development and treatment of glioma cells. The main tumor-associated macrophages (TAMs) are the M1-polarized subtype with proinflammatory and antitumoral functions and the M2-polarized subtype with anti-inflammatory and protumoral effects [123–125]. The M2 subtype plays a vital role in the TIME by excreting extracellular matrix (ECM) components, promoting T-cell anergy and stimulating angiogenesis [126,127]. In addition, TAMs may increase the tumorigenicity and chemoresistance of glioma cells by revising the stromal and blood vessel architecture [128]. Myeloid-derived suppressor cells (MDSCs), as myeloid-derived progenitor cells that accumulate in the TIME of glioma, suppress the proliferation and activity of T cells by releasing inducible nitric oxide synthase, ROS, cyclooxygenase-2 and transforming growth factor-β [129,130]. The regulatory T (Treg) cells accumulating in the TIME of glioma could inhibit immune surveillance and attack by excreting IL-10, IL-35 and TGF-β, which possibly predicts the poor prognosis of patients with gliomas [131–133]. Neutrophils are able to not only inhibit the antiangiogenic therapy of other tumors, but also predict the poor prognosis of patients with gliomas [134].

**Figure 3.** Role of ferroptosis in glioma immunity. (**a**) CD8+ T cells release IFNγ to activate INFR (which inhibits SLC7A11 transcription through STAT1) to promote tumour cell ferroptosis. TGFβ1 released by macrophages induces the downregulation of system xCT mediated by SMAD proteins, thereby triggering lipid ROS-mediated ferroptosis via the GSH-GPX4 axis. In turn, ferroptotic glioma cells release DAMPs (such as HMGB1 and AA) to promote the recruitment and activation of immune cells. (**b**) In contrast, DAMPs, such as HMGB1, KRAS-G12D and 8-OHG, could affect the function of macrophages in the tumour microenvironment. In particular, KRAS-G12D binds AGER on the cell surface of macrophages to trigger M2 macrophage polarization, which might limit antitumour immunity. IFN-γ, interferon-γ; INFR, interferon receptor; STAT1, signal transducer and activator of transcription 1; GSH, glutathione; GPX4, glutathione peroxidase 4; DAMPs, damageassociated molecular patterns; HMGB1, high mobility group protein B1; AA, arachidonic acid; 8-OHG, 8-hydroxyguanosine; AGER, advanced glycosylation end productspecific receptor.

However, the regulation of ferroptosis in the TIME appears to show the treatment limitations of glioma. Due to the reducing supply of oxygen and nutrients, as well as promoting acidosis in the TIME [135,136], glioma cells will survive due to their own tremendous plasticity [137–139], but immune cells cannot adapt and lose the effect [140,141]. In addition, the function of immune cells could be inhibited due to the cytokines secreted by glioma cells and immune cells, which may express ferroptosis [142,143]. The Treg cells that impede immune surveillance of tumors, an immunosuppressive subset of CD4+ T cells, are resistant to ferroptosis, which is likely due to GPX4 induction in activated Treg cells [144]. A study has shown that KRAS-G12D can be released into exosomes by pancreatic cancer cells upon ferroptosis and taken up by macrophages via advanced glycation end products (AGEs), which ultimately stimulate tumor growth through the polarization of macrophages to the M2 phenotype. In addition, conditional deletion of Gpx4 in the pancreas of mice promoted mutant Kras-driven tumorigenesis through ferroptotic injury-induced DNA release and subsequent STING-activated inflammation in macrophages [12]. Previous experiments and database analysis showed that the infiltration of Treg cells, neutrophils, and M2-polarized subtype macrophages in the TIME was significantly increased at high levels of ferroptosis [121]. Similarly, MDSCs with immunosuppressive functions exhibit resistance to ferroptosis due to an inhibitory drive on the p53-heme oxygenase 1 (HMOX1) axis mediated by N-acylsphingosine aminohydrolase 2 (ASAH2). In addition, large amounts of lipid peroxides were detected in tumor-derived CD8<sup>+</sup> T cells, but not in lymph nodederived CD8+ T cells, which suggested that ferroptosis may be the metabolic vulnerability

of tumor-specific CD8+ T cells. In addition, ferroptosis inducers (especially GPX4 inhibitors) may reduce antitumour immunity and promote tumor development by impairing CD8<sup>+</sup> T cells and follicular helper T cells (Tfhs) [144]. Under these conditions, ferroptosis may promote tumor growth by suppressing antitumour immunity. These advances indicate that ferroptosis has great potential to enhance the immunotherapy in cancer treatment [145,146].

#### **4. Challenges of Ferroptosis in Glioma**

While ferroptosis has a significant effect on the growth and treatment of gliomas, the mechanism needs to be further explored [147]. Most RCD effector molecules are proteases or porins, for example, caspases as well as mixed lineage kinase domain-like protein (MLKL) are involved in apoptosis and necrosis, respectively, and GasderminD participates in pyroptosis [148–150]. PLOOH is currently regarded as the ultimate executor of ferroptosis; however, whether effector molecules still exist downstream of PLOOH requires more exploration. [145]. Cytoplasmic GPX4 detoxifying lipid peroxides accumulated at the plasma membrane were unable to inhibit mitochondrial lipid peroxidation-induced ferroptosis in GPX4 knockout cells treated with DHODH inhibitors, which indicated that additional ferroptosis enforcement mechanisms may exist downstream of cytoplasmic lipid peroxidation [144]. The interaction between ferroptosis and other RCDs is also unclear. Some features of ferroptosis compared to other RCDs are not unique; for example, ferroptosis signals such as lipid peroxidation and regulators such as GPX4 and SLC7A11 can also regulate other types of RCDs. Ferroptosis may also modulate the TIME by interacting with other RCDs, thereby affecting the development and treatment of gliomas. In addition, ferroptosis currently has limitations in the diagnosis and treatment of gliomas. Currently, some targets, especially GPX4, ACSL4, P53 and FTH1 [151,152], are regarded as biomarkers, but they are not still a gold standard. Some significant biomarkers that can accurately predict the tumor response to ferroptosis induction are urgently needed, especially those that can be detected directly in patient blood, urine, feces and tumor tissue. It is also not clear what types of patients with gliomas are more sensitive to ferroptosis treatments. Three criteria, including iron levels, gene expression and mutations, can be combined to assess which patients are most likely to benefit from ferroptosis; for example, SLC7A11 inhibitors may be particularly effective against certain types of gliomas that overexpress this target. Finally, ferroptosis plays a dual role in the development and treatment of tumors. Ferroptosis promoted glioma cell death, but also reduced the treatment effect of gliomas by enhancing the levels of Treg cells, neutrophils, and M2-polarized subtype macrophages in the TIME to suppress antitumour immunity [121]. Tumor cells sacrificing themselves could also get the surrounding tumor cells to be in a stress state and finally avoid ferroptosis by secreting cytokines [145]. What substances are released by tumor cells after ferroptosis and the effects of these signals need to be further studied [153,154]. More evidence is needed to confirm whether the cytokines released by cells after ferroptosis enable surrounding glioma cells to evade immune surveillance by regulating TIME [155,156].

#### **5. Conclusions and Future Perspectives**

Ferroptosis, as a new programmed cell death mode, is different from other RCDs and is the result of iron-dependent lipid peroxidation accumulation. In this review, we focused on the regulatory mechanism of ferroptosis and found that ferroptosis plays multiple roles in the occurrence and development of glioma. Ferroptosis not only induces glioma cell death but also promotes glioma cell growth, invasion, migration, and resistance by regulating TAMs, MDSCs, Treg cells, neutrophils and CD8+ T cells in the TIME. Notably, studies have shown that some new compounds (such as strychnine, dihydroartemisinin and ibuprofen) are capable of inducing ferroptosis in gliomas, and ferroptosis-induced chemosensitizers, including erastin, can be used in combination with various drugs (such as cisplatin, temozolomide and cytarabine), which may provide new therapeutic opportunities for glioma treatment. However, poor BBB penetration reduces targeting tumor ability, and potential compensatory mechanisms hinder the effectiveness of ferroptosis agents in

glioma therapy. It has been suggested that designing nanoengineered systems to improve the targeted delivery of drugs can overcome these issues, which further enhances the effectiveness of glioma treatment [157–159] (Table 2). Overall, although ferroptosis has great advantages in glioma treatment, we still need multidisciplinary cooperation to further explore the pros and cons of targeting ferroptosis and to evaluate its potential value in clinical applications.

**Table 2.** Various NP-based drug delivery systems for the potential treatment of glioma.


**Author Contributions:** Conceptualization, Y.P., G.Y. and Y.L.; investigation, Y.L. and G.T.; writingoriginal draft preparation, Y.L.; writing-review and editing, Y.L. and X.F.; visualization, Y.L. and S.B.; supervision, Y.P. and G.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by grants from the National Natural Science Foundation of China (81960541/82060455).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**


71


#### **References**


29. Gao, G.; Li, J.; Zhang, Y.; Chang, Y.Z. Cellular Iron Metabolism and Regulation. *Adv. Exp. Med. Biol.* **2019**, *1173*, 21–32. [CrossRef]

30. Yan, H.F.; Zou, T.; Tuo, Q.Z.; Xu, S.; Li, H.; Belaidi, A.A.; Lei, P. Ferroptosis: Mechanisms and links with diseases. *Signal Transduct. Target. Ther.* **2021**, *6*, 49. [CrossRef]


**Bruce E. Holbein <sup>1</sup> and Christian Lehmann 2,\***


**Abstract:** Iron is irreplaceably required for animal and human cells as it provides the activity center for a wide variety of essential enzymes needed for energy production, nucleic acid synthesis, carbon metabolism and cellular defense. However, iron is toxic when present in excess and its uptake and storage must, therefore, be tightly regulated to avoid damage. A growing body of evidence indicates that iron dysregulation leading to excess quantities of free reactive iron is responsible for a wide range of otherwise discrete diseases. Iron excess can promote proliferative diseases such as infections and cancer by supplying iron to pathogens or cancer cells. Toxicity from reactive iron plays roles in the pathogenesis of various metabolic, neurological and inflammatory diseases. Interestingly, a common underlying aspect of these conditions is availability of excess reactive iron. This underpinning aspect provides a potential new therapeutic avenue. Existing hematologically used iron chelators to take up excess iron have shown serious limitations for use but new purpose-designed chelators in development show promise for suppressing microbial pathogen and cancer cell growth, and also for relieving iron-induced toxicity in neurological and other diseases. Hepcidin and hepcidin agonists are also showing promise for relieving iron dysregulation. Harnessing iron-driven reactive oxygen species (ROS) generation with ferroptosis has shown promise for selective destruction of cancer cells. We review biological iron requirements, iron regulation and the nature of iron dysregulation in various diseases. Current results pertaining to potential new therapies are also reviewed.

**Keywords:** iron homeostasis; iron dysregulation; inflammation; infection; cancer; iron chelation

### **1. Introduction**

When oxygen first appeared in the Earth's atmosphere, iron with its Fe2/Fe3 redox potential of 0.771 V represented a sweet spot for chemical catalysis of a wide range of oxidation and reduction reactions needed for biosynthesis and energy production by all living organisms, except for a few microbes. Around 2% of human genes encode for iron proteins, with more than half of these having catalytic function, with oxidoreductase class enzymes being the largest fraction [1].

#### *1.1. Iron as Biological Catalyst*

Table 1 provides examples of key iron-dependent enzymes needed by most cells for critical functions at a number of physiological levels. These enzymes are critically important for cell growth and replication and generally, iron cannot be substituted for activity in these. This underlies the irreplaceable need for iron by all higher organisms.

**Citation:** Holbein, B.E.; Lehmann, C. Dysregulated Iron Homeostasis as Common Disease Etiology and Promising Therapeutic Target. *Antioxidants* **2023**, *12*, 671. https:// doi.org/10.3390/antiox12030671

Academic Editor: Yan-Zhong Chang

Received: 15 February 2023 Revised: 2 March 2023 Accepted: 7 March 2023 Published: 9 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).


**Table 1.** Iron-dependent enzymes.

#### *1.2. Excess Iron Toxicity*

Seemingly paradoxical, iron is also toxic to cells because of its production of oxidizing radicals with their formation driven by redox cycling of iron. Superoxide (O2 −) is produced constitutively by the electron transport chain in cells by slippage of an electron to O2 with concomitant reduction of Fe3 to Fe2. The human body produces around5gO2 −/day [9], but superoxide dismutase (an iron dependent enzyme) normally neutralizes quantities in excess of normal amounts as needed for cell regulation and signaling [10]. Superoxide is also produced by nicotinamide adenine phosphate oxidase in the phagocytic defensive cells of vertebrate animals and this is used to kill phagocytosed invading microbes [11]. Superoxide can set up a cascade cycling of Fe reduction/oxidation coupled to peroxide and hydroxyl radical ·OH production, as shown in Scheme 1, with ·OH being highly toxic through damage to DNA and membrane lipids [12].

**Scheme 1.** Reactive oxygen species generation.

#### *1.3. Chemical Containment*

Containment of reactive iron and its ROS products is achieved through both compartmentalization and its chemical chaperoning. Phagocytes, including macrophages and polymorphonuclear leukocytes, compartmentalize ROS production within intracellular phagosomes, which spares other intracellular organelles and the extracellular milieu from direct ROS exposure [13]. The bulk of total body iron stores represents around 55 mg/kg and is located intracellularly, safely incorporated within hemoglobin (erythrocytes), myoglobin (muscle cells) and ferritin (liver cells) [14]. Iron incorporated into heme and ferritin is not freely chemically available to participate in ROS production. Body iron stores are shuttled to sites of use in other cells by transferrin, which binds iron avidly so that it is effectively chaperoned in circulation and not available for ROS reactions. There is an additional small labile iron pool located both intracellularly and extracellularly which is readily ROS-reactive [15], and this pool is important in relation to iron dysregulation, which increases the amounts in this labile pool.

It is interesting to note that some currently used therapeutic agents, including aminoglycoside antibiotics, can bind iron not fully satisfying its chaperoning requirements [16] and can induce ROS-related tissue damage [17], likely due to the mobilization of labile iron. This underscores the need for containment and chaperoning of labile ROS-reactive iron in the host as part of normal homeostatic control.

#### **2. Regulation of Normal Iron Homeostasis**

Of the 3750 mg of total iron, around 65% of this is incorporated into heme in erythrocytes, 10% within heme of myoglobin of the muscles, 14% in macrophage cells of the reticuloendothelial system (RES), 28% stored within ferritin in hepatocytes and 4% in bone marrow cells [14]. Iron taken up by enterocytes in the gut is stored transiently in small amounts in ferritin until there is demand for iron replenishment elsewhere in the body and then transferrin takes up this iron into the bloodstream, serving as a shuttle/delivery protein chaperone. The transferrin pool is normally only 30% saturated with iron and, therefore, it provides iron-holding capacity to limit amounts of labile non-transferrin-bound iron in circulation. The transferrin iron pool is highly dynamic and represents a flux of around 25 mg iron per day (Pantopoulos, 2018—see Figure 1) [18]. The body has no excretory mechanism for iron and small daily losses of 1–2 mg from skin desquamation and other sources are correspondingly compensated by uptake from the gut.

**Figure 1.** Dynamics of systemic iron balance. Plasma transferrin delivers iron to bone marrow erythroblasts and to other tissues. It contains a very small (∼0.1%) but highly dynamic fraction of body iron that turns over >10 times/day to meet the iron need for erythropoiesis (20–30 mg/day). The transferrin iron pool is primarily replenished with iron recycled from hepatic and splenic macrophages during erythrophagocytosis of senescent red blood cells. Duodenal enterocytes absorb dietary iron and release small amounts (1–2 mg/day) to compensate for non-specific losses. Hepatocytes store excess of body iron, which can be mobilized to plasma under iron deficiency. Iron efflux to plasma from macrophages, enterocytes or hepatocytes is negatively regulated by hepcidin, a liverderived peptide hormone that binds to the iron exporter ferroportin and promotes its degradation (Pantopoulos, 2018—with permission) [18].

Hepcidin, a 25-amino acid peptide hormone produced primarily by liver hepatocytes, is the master regulator of iron homeostasis and it negatively regulates iron uptake by inactivating plasma membrane-bound ferroportin on enterocyte and macrophage cell membranes as needed for transfer of intracellular iron to extracellular compartments [19]. This provides tight regulation of iron homeostasis, which is critical for ensuring no excess toxic quantities of iron either intracellularly or extracellularly. Iron dysregulation often presents with elevated amounts of circulating plasma iron including transferrin-bound iron with >50% saturation along with elevated non-transferrin-bound iron (NTBI) that can reach >0.5 μM [20]. It is the labile portion of NTBI that plays a role in the pathogenesis of the various non-microbial diseases we review below, while elevated plasma iron in the form of transferrin-bound iron can also support microbial infection [21].

#### **3. Iron Dysregulation Underlying Disease**

#### *3.1. The Nature of Iron Dysregulation*

Iron dysregulation diseases often result from increased amounts of circulating labile reactive plasma iron. This can be evidenced by elevated plasma transferrin-bound iron (TBI) with transferrin saturation above its normal 30%, sometimes reaching 100% saturation [22]. With high saturation of transferrin, some of the plasma iron is not effectively chaperoned and this provides a pool of non-transferrin-bound iron (NTBI) labile reactive iron (LPI). This LPI is also more mobile and capable of entering cells, where it is toxic [23,24].

Excess plasma iron in the form of TBI and NTBI LPI influences two main categories of diseases. Elevated plasma iron, especially in the form of transferrin iron, supports cell-proliferative diseases including infection and cancer. Increased plasma iron present as non-transferrin-bound LPI is part of the pathology of other diseases where excess reactive iron triggers iron-related pathologies. The various diseases we review are summarized in Table 2.


**Table 2.** Diseases with iron dysregulation.

#### *3.2. Infections*

All pathogenic microorganisms, with the exception of *Borrelia burgdorferi*, the agent of Lyme disease, have absolute and irreplaceable requirements for iron for their growth and replication in our bodies [37]. *B. burgdorferi* can utilize manganese in place of iron [38]. The vertebrate host normally maintains conditions of low iron bioavailability to microbes, especially in extracellular compartments such as plasma, respiratory secretions and tears where infection is often initiated, thus providing a natural nutritional immunity. Transferrin in plasma and lactoferrin in tears and other secretions maintain very low levels of freely available iron [39]. Lactoferrin concentrations can reach >3 mg/mL in tears [40]. Furthermore, when infection is first detected, the body mounts an early iron withdrawal defense response, where extracellular iron concentrations are further reduced by moving

this iron to intracellular stores [19]. Successful pathogens deploy a variety of virulence mechanisms to successfully compete for iron, as reviewed elsewhere [21].

#### 3.2.1. Pathogenic Microbes

Iron has now been found to support infection by almost all pathogenic microbes and Table 3 summarizes some important microbial infections in relation to the main host iron sources feeding the infection. Of these, some have cell-wall surface receptors to intercept host iron sources, while others deploy high-affinity siderophores to effectively strip iron from host transferrin or lactoferrin [21].

#### **Table 3.** Iron sources for pathogenic microbes.


Interfering with bacterial iron acquisition provides a new therapeutic avenue to fight infection, as discussed later in this review.

#### 3.2.2. Bacterial Sepsis

Sepsis is a life-threatening condition killing around 11 million people worldwide annually [51]. It results from a severely dysregulated host response to inflammation. While sepsis can develop form viral infections such as COVID-19 [51], it most often develops through microbial infection. Bacterial sepsis can develop from serious bacterial infection with the host inflammatory response overstimulated and becoming dysregulated [52], with this often triggered by bacterial cellular components released during infection, including endotoxin [53].

During sepsis, iron metabolism is altered with increased iron uptake into cells and this has been associated with increased iron-driven oxidative injury and cell death [52]. High serum iron levels are also associated with sepsis and poor sepsis outcomes [54]. In fact, transferrin saturation reflecting serum iron availability is strongly correlated to sepsis outcomes, with increased iron availability pronounced in lethally ill patients [54,55]. This aspect, as driven by excess iron, is additional to iron feeding microbial infection, as it relates to host dysregulated inflammation. Thus, the condition of iron dysregulation provides for perfect storm conditions of increased iron availability to support rapidly proliferating microbes on the one hand and increased host damage from the associated dysregulated inflammatory response on the other hand.

Sepsis therapy presents a large unmet medical need with no currently approved therapeutics available for clinical use. However, the underlying iron dysregulation provides a new therapeutic avenue for serious infection/sepsis, as discussed later in this review.

#### 3.2.3. Parasitic Pathogens

Multicellular parasitic organisms also require iron to invade and mount infection in vertebrate hosts [26]. Leishmania chagasi, which causes leishmaniasis, can utilize iron from transferrin, lactoferrin or heme when growing in its promastigote form, taking these up directly without deployment of iron-intercepting siderophores [56]. Trypanosoma brucei employs transferrin surface receptors and Entamoeba histolytica employs lactoferrin receptors for iron acquisition [26]. Plasmodium falciparum, which causes malaria, has also been shown to possess surface receptors for transferrin iron, which is interesting in that it resides

within hemoglobin-rich erythrocytes [57]. Addition of iron has been shown to promote infection with Trypanosoma cruzi [58] and iron supplementation has been associated with increased risk of malaria [57]. Parasitic infection, in contrast to most microbial infections, is chronic in nature and has been associated with what is described as the anemia of chronic disease [59]. This also occurs in cancer, as further discussed later, and this chronic anemia is considered the result of the prolonged effort of the host to restrict iron to the invading pathogens or cancer cells. Interestingly, host iron deficiency has been found to be protective against malaria, based on epidemiological studies [57].

#### 3.2.4. Viral Infections

Viruses have no direct requirements for iron, but it is now evident that viral replication in host cells is affected by host iron in the case of both DNA viruses, such as hepatitis B (HBV) and human cytomegalovirus (HCMV), and RNA viruses, such as hepatitis C (HCV) and human immunodeficiency virus (HIV) [27]. It remains somewhat obscure as to whether iron dysregulation predisposes the host to these viral infections, or alternatively, if the viral infection leads to host iron dysregulation. Elevated iron levels are associated with the progression of chronic HBV infection [60] and iron addition has been shown to enhance HCV viral replication in vitro [61]. Transferrin receptor-1 mRNA levels were increased due to HIV infection, leading to an increased iron uptake and higher level of cellular iron [62]. However, it has been suggested that in cirrhotic patients, HBV-related liver injury, but not the HBV infection itself, may be cause changes in serum iron markers [63].

#### *3.3. Cancer*

The role of iron and its dysregulation in cancer is multi-faceted, as reviewed by Torti et al. (2018) [64]. Stevens et al. (1994) reported on a cohort of 14,000 US National Health and Nutrition survey participants with a major finding that participants with higher transferrin (Tf) iron saturation levels were at higher risk of cancer than participants with lower transferrin Tf saturation levels, a finding supported by subsequent studies [65]. Cancer cells arising from various tissues such as breast or liver have increased need for iron compared to normal cells and produce increased amounts of cell-surface transferrin receptors in response to their increased and continuous iron needs [66]. Targeting transferrin receptors has been proposed for its therapeutic potential [67] as shown using antibodies directed to cancer-cell-surface transferrin receptor TfR1 [68]. This aspect has great appeal as common features to all cancers are possession of TfR1 receptors [68], their altered iron metabolism and increased needs for iron to support their rapid growth [69] and their primary dependence on host transferrin as the source of this iron. Thus, any advances in regard to beneficial iron restriction could apply to various cancers broadly.

Cancer also induces a chronic anemia, as seen in other chronic diseases, and this appears to be a host attempt to restrict cancer growth [64]. Somewhat paradoxically, cancer cells are more susceptible to ferroptosis, a programmed cell death triggered by excess reactive iron [70], suggesting host iron withdrawal defense to restrict cancer growth could also impede cancer cell killing via ferroptosis.

#### *3.4. Ferroptotic Cell Death*

Ferroptosis is an additional form of regulated cell death (RCD), but unlike other forms such as apoptosis and necrosis, it is caused by an iron-dependent accumulation of lipid peroxides which kill cells, although it shares other common features with the other modes of RCD [70]. ROS produced through the iron-catalyzed Fenton reaction contribute to its initiation [71] and, therefore, iron dysregulation with its excess labile iron is a predisposing factor.

Potential inhibitors of ferroptosis have been investigated including ROS-trapping antioxidants, such as α-tocopherol and ferrostatin-1, and these have shown potential for reducing ferroptotic damage [72].

Excess labile iron driving ferroptosis has also been demonstrated through its suppression by addition of the iron chelator deferoxamine [73]. However, ferric nitriloacetate (NTA) has been shown to promote ferroptosis and renal carcinoma by driving Fenton activity [71]. It is important to note that various iron chelators differ in their abilities to fully coordinate and, therefore, fully chaperone iron [21]. Deferoxamine can fully hexadentate-coordinate iron within a single deferoxamine molecule, while NTA requires two chelator molecules to fully satisfy a single iron atom. Therefore, depending on the prevailing chelator and iron concentrations, incompletely coordinated, Fenton-reactive species can be formed, a problem which appears to underlie the toxicity of some of the currently used medical chelators [21]. This problem of small molecule cell-permeable chelators is compounded in that they can more readily penetrate cells, reaching cellular iron stores and, therefore, mobilize additional labile iron supplies, this in turn exacerbating iron dysregulation.

The excess supply of labile reactive iron observed as a feature of iron dysregulation appears to underpin tissue toxicity with a number of diseases and ferroptosis appears to be at least part of the pathology of these diseases, as further discussed below.

#### *3.5. Inflammatory Diseases*

While inflammation typically accompanies infection and cancer, other diseases appear to be primarily inflammatory in nature and iron dysregulation and its associated ROS activity are involved in the inflammatory response.

#### 3.5.1. Ocular

Iron dysregulation of the eye has been linked to various eye diseases affecting the cornea and retina, including corneal epithelial disease, corneal endothelial cell dysfunction, retinal pigment epithelial (RPE)-associated eye diseases, glaucoma, diabetic retinopathy (DR), retinal ischemia/reperfusion injury (RIRI), retinoblastoma, retinitis pigmentosa (RP) and age-related cataracts, and common to these, ferroptosis pathology has been implicated [74]. Ferroptotic iron-induced toxicity through ROS damage has been directly linked to corneal diseases such as cataractogenesis, inflammatory retinal diseases such as agerelated macular degeneration (AMD) and optic neuropathies [35]. Transferrin mRNA levels are also upregulated in AMD, possibly in response to increased retinal iron loads [75]. This feature provides a potential avenue to new therapeutics, including the use of iron chelators to treat eye diseases that are in urgent need of new therapeutics.

#### 3.5.2. Lung Fibrosis

Pulmonary iron content is also tightly regulated, as excess iron can catalyze ROS formation, and this has been linked to the pathogenesis of chronic inflammatory lung diseases such as idiopathic pulmonary fibrosis [32]. Iron accumulation was increased in lung sections from patients with IPF, and human lung fibroblasts show greater proliferation and cytokine and extracellular matrix responses when exposed to increased iron levels [76]. These authors provided direct evidence for iron overload affecting the progression of pulmonary fibrosis. They investigated whether changes in iron homeostasis are the cause or a consequence of pulmonary fibrosis and used mouse models of iron overload to show that iron accumulation results in impaired lung function and subsequently worse pulmonary fibrosis upon lung injury by bleomycin [76]. Lung fibrosis is a progressive irreversible disease, as fibrotic lung tissue does not repair/remodel, making anti-fibrotic agents an urgent need. Overcoming iron dysregulation to reduce iron-driven fibrosis is a new potential avenue for therapy. Iron chelation therapeutics have potential for use in slowing or stopping pulmonary fibrosis.

#### 3.5.3. Kidney

Increased kidney proximal tubule (PT) cell cytosolic non-transferrin Tf-bound, i.e., labile iron has been shown to induce the generation of ROS in PT cells and this could contribute to the progression of proteinuric chronic kidney diseases [34]. Recent clinical

studies using anti-oxidative drugs that serve to reduce labile iron suggest that chelation of iron in the kidney has beneficial effects on the course of chronic kidney disease [77]. In nephritic syndromes associated with damage to the glomerular filter, urinary Tf concentration dramatically increases and may even lead to hypo-transferrinemia, iron loss and microcytic anemia [78].

Nephritic kidney damage with urinary transferrin excretion is also part of the pathology associated with autoimmune diseases including lupus erythematosus [79] and rheumatoid arthritis [80], as further discussed further below.

#### *3.6. Diabetes*

High dietary iron and dysregulated iron metabolism are risk factors for type 2 diabetes mellitus (T2DM), affecting most of its features of decreased insulin secretion, insulin resistance and increased hepatic gluconeogenesis [29]. Dysregulated iron metabolism with increased serum levels of ferritin have been found in newly diagnosed type 2 diabetes but not in individuals with pre-diabetes [81]. Oxidative stress from ROS is now known to be a major underlying mediator of diabetic complications [82]. Iron and ferroptosis have been shown to participate in pancreatic beta cell death [83]. In model studies using T2DM, mice insulin secretion was worsened by ferroptosis-inducing compounds. However, quercetin (a natural iron chelator), ferroptosis inhibitor ferrostatin-1 and iron-chelating deferoxamine, each rescued cell viability when cells were challenged with high glucose [84]. These studies support the potential for use of iron-chelating therapeutics in the treatment of T2DM.

#### *3.7. Cardiovascular*

Ferroptosis driven by iron dysregulation has now been implicated in several cardiovascular disease conditions including cardiomyopathy, atherosclerotic disease and myocardial ischemia/perfusion injury [30,85]. Inhibition of ferroptosis by ferrostatin-1 improved cardiac function and reduced mortality in a doxorubicin-induced mouse model of cardiomyopathy, which was associated with the release of free cellular iron caused by HO-1 upregulation [86]. However, as pointed out by Li and Zhang (2021), the mechanisms of ferroptosis in heart and vasculature disease remain elusive [30].

#### *3.8. Autoimmune*

The role of iron regulation in immune-related diseases was recently reviewed by Cronin et al. (2019) [87]. Substantial evidence has now linked iron dysregulation to the pathogenesis of lupus erythematosus [88]. Importantly, the common serious complication, lupus nephritis, has been linked to renal iron accumulation [79] and ferroptosis kidney cell damage has been described as an important feature of its pathology [88].

#### *3.9. Neurological*

Many neurological conditions have been shown to coincide with altered bodily distribution of various transition series biometals, especially in the case of iron [89]. Iron dysregulation with increased labile plasma iron supply and resulting increased ROS, creating oxidative stress and damage on neurological tissues, has now been linked to Friedreich's ataxia, Alzheimer's disease (AD), Parkinson's disease, Multiple Sclerosis (MS) and Amyotrophic Lateral Sclerosis (ALS) [31]. Increased ferroptosis with corresponding destruction of neurological cells has been described in Parkinson's disease and MS [90] as well as for Alzheimer's disease [91].

For example, Parkinson's disease (PD) is characterized by progressive motor impairment attributed to progressive loss of dopaminergic neurons in the substantia nigra (SN) pars compacta. In addition to an accumulation of iron, there is also an increased production of reactive oxygen/nitrogen species (ROS/RNS) and inflammatory markers seen in this pathology [92]. In addition, abnormal iron increases are commonly detected in AD patients, although controversy continues regarding iron's association with AD plaques [91]. It now

appears that a common feature of iron dysregulation underlying neurological diseases is iron-driven ferroptosis neurological cell death [93].

#### *3.10. Iron Overload*

While body iron overload can follow repeated blood transfusions, as seen in patients with Thalassemia, i.e., because the body lacks an excretory pathway for excess iron, congenital disorders of iron overload such as hemochromatosis represent iron overload disorders caused by dysregulated iron homeostasis. In hemochromatosis, disruption of the hepcidin pathway due to mutations in genes encoding auxiliary factors in iron signaling to hepcidin result in insufficient hepcidin responses to iron intake or to high body iron stores. This causes loss of hepcidin-mediated feedback inhibition in dietary iron absorption and consequently unregulated uptake of dietary iron, elevated transferrin iron saturation and appearance of labile reactive non-transferrin-bound iron [18].

#### *3.11. Cirrhosis*

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, beginning with the presence of >5% excessive lipid accumulation in the liver, and typically developing into non-alcoholic steatohepatitis, fibrosis, cirrhosis and often hepatocellular carcinoma. Excess free reactive iron and its associated ROS-mediated tissue damage have been positively associated with severity of NAFLD [36].

#### *3.12. Anemia of Chronic Infection and Inflammation*

The anemia of inflammation (AI), often referred to as the anemia of chronic disease (ACD), is a secondary anemia that often develops slowly as a result of chronic inflammation during chronic infection (especially parasitic), cancer and with at least some of the other inflammatory diseases we have reviewed, such as diabetes and autoimmune diseases [59,94,95]. Overall, it appears to be a host defensive mechanism. Interestingly, a short-term hypoferremia response is often seen early in the acute stages of infection and this has also been shown to be triggered by inflammatory mediators such as ILK-6 and demonstrated to be a relatively short-term active mechanism for restricting iron supply to growing invaders [21]. The diagnostic challenge in AI/ACD is the identification of patients with concomitant true iron deficiency because they need specific evaluation for the source of blood loss and iron-targeted management strategies [Weiss et al. 2019].

#### **4. Therapeutic Options**

#### *4.1. Iron Restriction*

Higher heme iron intake and increased body iron stores were significantly associated with a greater risk of type 2 diabetes, as shown in a meta-analysis of 11 prospective studies [96]. Interestingly, blood-letting to reduce overall body iron stores has shown potential benefit in the treatment of diabetes [97].

In principle, restriction of iron uptake would also lower amounts of deleterious liable reactive iron. However, withholding (or supplying) iron has remained controversial in medicine over the years due to the delicate balance required for iron homeostasis and the consequences of triggering anemia. While supply of dietary iron would be safer, administration of parenteral iron to treat anemia, especially the anemia of chronic disease, has serious implications. Given the roles of dysregulated iron in infection and other diseases, this requires careful consideration and likely should be avoided if possible.

#### *4.2. Iron Chelators*

Iron chelators that bind and can sequester excess available labile reactive iron have shown potential for the therapy of several diseases associated with iron dysregulation. However, medical chelators to date have been developed to treat hematological conditions associated with high excess body iron stores (e.g., transfusional iron overload in Thalassemia patients) but were not developed as iron dysregulation therapeutics. We have

provided a number of important potential attributes for ideal iron dysregulation chelator therapeutics, as summarized in Table 4.

**Table 4.** Iron chelators.


<sup>a</sup> Holbein, unpublished CRO report: JBL-DMPK-0062-0016, <sup>b</sup> Holbein, unpublished CRO reports: SP-JBL/NG-RIVR-142 and JBL/NG-ROR-052.

A number of clinical trials or natural products with iron-chelating properties, such as curcumin and polyphenolics, have shown promise for lowering excess iron [105] but none have been approved for clinical use.

#### *4.3. Hepcidin and Agonists*

Hepcidin mimetics, stimulators of its production and ferroportin inhibitors have seen early clinical stage testing as to their safety and potential efficacy [106]. The ferroportin inhibitor VIT-2763 has advanced to phase II trials and it has shown both low toxicity and good potential for lowering serum iron levels [107] Other approaches of using hepcidin mimetics including PTG-300 (rusfertide) have shown promising phase III results. As of yet, none of these have received regulatory approval for ongoing clinical use [108].

#### **5. Future Needs and Conclusions**

We identified a number of gaps in our current understanding of iron dysregulation in the pathology of diseases. A better understanding of the role of Fe dysregulation mechanisms for various diseases, especially related to cause or effect, is needed. There is a particular need for further understanding of anemia of chronic disease. Possible targeted ferroptosis induction in cancer can be considered once its mechanisms are elucidated.

A number of conclusions can be drawn at this stage. Therapeutics affecting iron dysregulation and lowering excess levels of labile reactive ion should be readily applicable to treating infection, including sepsis, and should also apply to cancer and other nonproliferative diseases from iron dysregulation. Improved chelators have immediate appeal as the broadest and preferred therapeutic approach.

Hepcidin mimetics and agonists have demonstrated potential and longer-term potential.

**Author Contributions:** Conceptualization, B.E.H.; writing—original draft preparation, B.E.H.; writing—review and editing, C.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors would like to thank Cassidy Scott for the help with the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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