Ferroptosis and its clinical significance

Introduction: Ferroptosis, mentioned as such for the first time in 2012 by Dixon et al., is an iron-dependent type of cell death that occurs in the presence of lipid peroxides. The mechanism of the process and the signaling pathways involved in it differ from the previously known apoptosis, necrosis, and autophagy. An inflammatory reaction also occurs, which further distinguishes this type of cell death from apoptosis. Ferrostatin inhibits this process.

Methods: A review of the literature obtained by searching the Medline database was performed, with a special focus on studies concerned with the importance of ferroptosis in clinical medicine, primarily hematology.

Results: Iron metabolism in malignant and healthy cells differs. Malignant cells tolerate oxidative stress well and avoid ferroptosis. According to literature data, various tested agents stimulate ferroptosis and thus become possible therapeutic agents. Some genes linked to iron metabolism have shown prognostic significance in patients with diffuse large B-cell lymphoma.

Conclusion: The discovery of new mechanisms of cell death and the signaling pathways involved in this process leads to potentially new target therapy. Although promising, these results require validation through further research.

INTRODUCTION

Ferroptosis, first mentioned in a study by Dixon et al. in 2012, is a non-apoptotic, non-necrotic, and non-autophagic form of cell death [1]. In 2003, Dolma et al. discovered that erastin can cause a new type of cell death, upon which Yagoda and Yang reported that iron chelators inhibited this process, which is based on mitochondrial dysfunction [2],[3],[4].

As the name suggests, ferroptosis is iron-dependent, it occurs in the presence of lipid peroxides, and is inhibited by ferrostatin. Unlike apoptosis, this process involves the involvement of inflammatory cells and an inflammatory reaction. In the process of apoptosis, the cell membrane swells and chromosomes condense. In ferroptosis, mitochondria are reduced, their membranes are hyperdense, and mitochondrial cristae are smaller or absent [1],[5],[6],[7],[8],[9]. In terms of biochemistry, the concentration of glutathione in the cell is lower. Glutathione peroxidase 4 (GPX4) is an enzyme that causes the reduction of lipid peroxides, and whose activity is also reduced in this process. The Fe² + ion oxidizes lipids, the concentration of reactive oxygen species (ROS) increases, and ferroptosis is triggered [5]. Transmembrane channels in cell membranes through which cystine and glutamate are exchanged are also important in this process. Cystine enters the cell and is reduced to cysteine, which is necessary for the synthesis of glutathione [1],[5]. This is also the site where ferroptosis inducers act. Iron chelators inhibit this process, but so do ferrostatin 1 (Fer-1), vitamin E, and liproxstatin 1.

Ferroptosis depends on iron, which is why it can be expected that factors involved in iron metabolism or in the regulation of its metabolism have either already proven or potential roles in the regulation of this type of cell death, making the cell more or less sensitive to ferroptosis [6],[10],[11].

Iron metabolism in healthy and malignant cells

When the regulation of iron metabolism is concerned, we speak of control at the cellular level and of systemic iron metabolism regulation. Iron homeostasis in the cell is regulated mainly by post-transcriptional control of genes via the interaction of iron-responsive elements (IRE) and iron-responsive proteins (IRP) [12].

Systemic homeostasis is controlled by hepcidin, a liver protein whose concentration, among other things, increases in inflammation, under the influence of interleukin-6, leading to the internalization and degradation of ferroportin. According to currently available literature data, ferroportin is the only exporter of iron from cells [12].

Dietary iron leaves the enterocytes via ferroportin, as trivalent iron, and is transported to tissues bound to transferrin. The iron-transferrin complex binds to the transferrin receptor 1 (TfR1) and enters cells via endocytosis. In the cell, trivalent iron is transformed into divalent iron and, as such, it is incorporated into various enzymes, it enters the composition of heme, and participates in oxidative phosphorylation and synthesis of deoxyribonucleotides. Excess iron is stored in ferritin.

It has been shown that two proteins – ZIP 8 and ZIP 14, with a known role in zinc transport, also participate in the transport of non-transferrin-bound iron (NTBI) through the cell membrane [12],[13],[14],[15],[16], and they are also important in hemochromatosis [16]. Their role in other conditions characterized by iron overload, such as malignancies and some forms of anemia, remains unclear [12]. Poly(rC) binding protein 1 (PCBP1) and poly(rC) binding protein (PCBP2) are iron chaperones – they accompany divalent iron in the cytoplasm and play a role in its distribution in the cell [12],[17],[18], while nuclear receptor coactivator 4 (NCOA4) participates in the autophagy of ferritin, thus affecting the utilization of iron stored in it [12],[19],[20],[21],[22].

The iron found in the cell deposited in ferritin is non-toxic, however, while bound in such a way, it is unavailable for cellular needs. For it to be usable, it must be released, usually by degradation in the lysosomes [19],[20],[21],[22]. It seems that the method of ferritin degradation is the same in conditions characterized by iron overload and those characterized by iron deficiency, however, it is delivered to lysosomes differently [12],[20]. When there is iron deficiency, autophagy occurs, and when there is iron overload, other pathways are active [20]. NCOA4 is an essential factor in ferritin autophagy in iron deficiency, as well as in the regulation of iron homeostasis in the spleen [23],[24]. If there is no iron deficiency, non-autophagic pathways of ferritin degradation are less active in malignant cells than in healthy ones, and therefore malignant cells are more resistant to damage caused by excess iron [20]. The reactivation of these pathways could increase the toxicity of iron to the neoplastic cell and potentially be a new therapeutic strategy [12].

Cancer cells alter the metabolism of thiols, which are intracellular redox buffers, and oxidative stress, due to excess iron, is associated with carcinogenesis [25]. We have already mentioned that malignant cells retain excess iron in lysosomes, but also in the cytoplasm and mitochondria. This leads to the disruption of their function, the uptake of glucose is increased, and, in the presence of oxygen, in the process of aerobic glycolysis, lactates are produced. This phenomenon is known as the Warburg effect. The property of higher uptake of glucose by malignant cells is used in the application of positron emission tomography (PET) in the diagnostics and assessment of the therapeutic effect of treatment in patients with malignant diseases [26].

Agents affecting ferroptosis

Based on all the above, it is evident that malignant cells, regardless of constant exposure to oxidative stress, can tolerate it and avoid ferroptosis [25]. Erastin and sulfasalazine block the entry of cystine through channels in the phospholipid part of the cell membrane. In this way, just like other agents affecting the reduced production of glutathione in experimental conditions, they lead to tumor growth suppression and ferroptosis [25],[27],[28],[29]. Tests have shown that change in the metabolism of cystine and glutathione leads to an increased concentration of free oxygen radicals, while antioxidant therapy prevents cell death [10]. Erastin is a strong inducer of ferroptosis that inhibits transmembrane antiporters for intracellular glutamate and extracellular cystine exchange. Glutathione participates in maintaining the redox balance and when its concentration is insufficient, free oxygen radicals accumulate, and it has already been said that cystine entry into the cell is necessary for its synthesis [30],[31],[32]. In their study, Dixon et al. reported that sulfasalazine and sorafenib blocked this antiporter leading to selective ferroptosis, however, erastin inhibited this system 2,500 times more potently than sulfasalazine. An additional indicator of the importance of iron in ferroptosis is that deferoxamine, an iron chelator, suppresses erastin-induced cell death, while additional iron intake promotes it [1],[27],[33]. Studies on diffuse large B-cell lymphoma cell cultures have shown significant sensitivity to erastin-induced cell death [34]. In another study, it was shown that erastin increased the sensitivity of leukemic cells in acute lymphocytic leukemia (ALL) to the effect of the second mitochondria-derived activator of caspases (SMAC) mimetic LCL-161 and that the synergistic action led to the death of leukemic cells. [35]. Other authors have reported a dose-dependent effect of erastin on acute myeloid leukemia cells, since, in their studies of cell cultures, it led to the suppression of their growth, but also to a mixed type of cell death. The suppression of this effect was achieved by ferrostatin 1, an inhibitor of ferroptosis, or necrostatin 1, an inhibitor of necroptosis, but not by an inhibitor of the caspase pathway, which acts on apoptosis, nor by chloroquine, which inhibits autophagy. These effects were not achieved in chronic myeloid leukemia, acute T lymphoblastic leukemia, and acute promyelocytic leukemia cell cultures. Acute myeloid leukemia (AML) cells were significantly more sensitive to cytosine arabinoside and anthracyclines [36] when low doses of erastin were administered, where doxorubicin and not daunoblastin was used. In a recent study, another group of authors demonstrated, in an in vitro model, that erastin increased the sensitivity of acute myeloid leukemia cells to venetoclax [37]. Fibroblasts and dendritic cells make cystine, which lymphoid cells depend on because they must absorb it in order to synthesize cystine. It has been demonstrated that sulfasalazine, acting on the system of channels through which cystine is transported in a culture of lymphoma cells, leads to the interruption of their proliferation, and later the suppression of tumor growth was confirmed in an animal model with transplanted lymphoma; there was no major harm to the host organism [38]. Induction of ferroptosis was achieved on a cell culture of 60 tumor lines by the action of artemisinin derivatives. Genes responsible for coding proteins involved in iron metabolism, such as transferrin, transferrin receptors 1 and 2, have been identified as predictors of sensitivity to artemisinin [39].

Ferroptosis in large B-cell lymphoma

Large B-cell lymphoma (LBCL) accounts for about a third of all non-Hodgkin’s lymphomas, which makes it the most common lymphoproliferative neoplasm in adult patients [40]. With first-line treatment, i.e. immunochemotherapy with the R-CHOP (rituximab-cyclophosphamide, doxorubicin, vincristine, prednisolone) regimen, complete remission is achieved in about 60% of patients, however, the treatment of patients with resistant or relapsing (R/R) disease remains a clinical problem. Not all patients with R/R LBCL are eligible for autologous stem cell transplantation, nor is chimeric antigen receptors (CAR) T-cell therapy widely available. Bispecific antibodies, combined with other drugs, are mostly available through clinical studies, however, it often happens that patients with R/R disease do not meet the criteria for inclusion in a study or that the studies are unavailable. Widely used standard risk scores are prognostic tools designed to estimate the probability of survival before and during the rituximab treatment era [41],[42]. Furthermore, LBCL is a heterogeneous disease represented by multiple entities in the new World Health Organization (WHO) classification [43], where there are sometimes overlapping interpretations and different views of the challenges, from the point of view of pathologists and clinicians [44]. New prognostic scores, as well as therapeutic agents that would act via other mechanisms, need to be developed.

Iron is necessary for many processes, including cell proliferation [45],[46]. Devin et al. developed a new score – iron score (IS), based on the expression profile of genes responsible for iron metabolism. In two independent cohorts of LBCL patients, which included 233 and 181 subjects, 11 genes showed prognostic significance. High expression of eight genes defined the poor prognosis group. IS was additionally validated in three cohorts in relation to overall survival (OS) [47]. IS was significantly higher in the activated B-cell-like (ABC) LBCL group, as compared to the germinal-center B-cell-like (GCB) LBCL group [47]. The same authors tested the effect of iron chelators deferasirox and deferoxamine on the viability of lymphoma cells on LBCL cell lines (separately ABC and GCB types) and, depending on the concentration, observed reduced cell viability after treatment with iron chelators. In the same study, the therapeutic effect of irinomycin was tested. It was found that nanomolar concentrations of irinomycin inhibited tumor cell growth. Iron supplementation inhibited the effect of chelators on lymphoma cells, but not the effect of irinomycin, suggesting a different action mechanism. Namely, iron chelators led to lymphoma cell apoptosis, while irinomycin led to iron sequestration in lysosomes, iron depletion in the cell cytosol, ferroportin degradation by autophagy in lysosomes, and the production of reactive oxygen radicals leading to ferroptosis [47].

In a study from 2021, Schmidt et al. showed that dimethyl fumarate, a drug approved for the treatment of multiple sclerosis and psoriasis, acted on both ABC LBCL and GCB LBCL cells by causing ferroptosis and weakening the NF-kB/STAT 3 signaling pathway, [48].

A year ago, a group of scientists from China announced that an elevated level of glutamine was associated with a poorer outcome in LBCL patients, and that the concentration of the glutamine derivative, αketo-glutarate, was inversely correlated with the aggressiveness of LBCL [49]. Accumulation of α-keto-glutarate led to oxidative stress in double-hit lymphoma (DHT). A high level of free oxygen radicals caused the accumulation of lipid peroxides, leading to the activation of TP53 and to ferroptosis [49].

CONCLUSION

There is an increasing number of studies dealing with ferroptosis in various malignancies, including hematological malignancies. The potential new roles of already-known drugs and the possibilities of their wider application and synergistic effect with standard therapy are being examined. The discovery of new cell death mechanisms and the signaling pathways involved in that process leads to the discovery of potentially new target therapies. From the clinician’s point of view, all this seems very interesting and promising, however, further clinical studies and validation of the results obtained in vitro and on laboratory animals are necessary.

NOTE: The basic principles of iron metabolism and ferroptosis were investigated in the doctoral dissertation of the first author of this study. The thesis was defended in 2022.