Introduction

The latest data released by the International Agency for Research on Cancer (IARC) indicates that there were 20 million new cancer cases globally in 2022, resulting in 9.7 million deaths. This data highlights the increasing global burden of cancer.1 However, the effectiveness of existing cancer treatments is concerning, as tumor cell resistance is reducing the efficacy of conventional therapies like radiotherapy and chemotherapy.2 Additionally, refractory cancers like small cell lung cancer (SCLC),3 triple-negative breast cancer (TNBC),4 and pancreatic cancer5 present significant challenges in oncology treatment due to their resistance to existing therapies. Ferroptosis, a novel iron-dependent form of programmed cell death first proposed by Dr. Brent R. Stockwell in 2012, is distinct from apoptosis, necrosis, and autophagy.6 It is triggered by glutathione (GSH) depletion, decreased activity of glutathione peroxidase 4 (GPX4), failure of lipid oxides to be metabolized by GPX4, and Fe2+-mediated oxidation of lipids leading to the generation of reactive oxygen species (ROS) that promote ferroptosis.7 Ferroptosis provides a new way of thinking about cancer treatment. Ferroptosis induction therapy has several natural advantages for cancer treatment. Firstly, the unique metabolism of cancer cells, the high load of ROS, and their specific mutations make some of them sensitive to ferroptosis,8 eg, clear cell renal cell carcinoma (ccRCC),9 pancreatic ductal adenocarcinoma,10 and TNBC.11 Cancer cells typically require more iron than normal cells to proliferate, and this iron dependence makes them more susceptible to ferroptosis.12 Cancer cells with a high mesenchymal state were found to be more resistant to a wide range of cancer treatments but were particularly susceptible to ferroptosis inducers.13 Also, ferroptosis induction therapy can be used in combination with conventional tumor treatments to enhance their therapeutic effects in certain malignancies.14

Ferroptosis-inducing therapies have emerged as a crucial strategy in oncology, with ongoing advancements in the development and study of various small molecule drugs targeting ferroptosis-regulating pathways.15 This review provides an overview of the regulatory mechanisms of ferroptosis and delves into the mechanisms of traditional small molecule ferroptosis inducers and their effects on various tumors. It also highlights the recent progress in inducing ferroptosis through innovative methods such as PROTACs, PDT, SDT, and nanomaterials. Furthermore, the review addresses the challenges and opportunities in developing ferroptosis-inducing agents, including the exploration of new targets, enhancement of selectivity, and mitigation of toxic and side effects (Figure 1).

Figure 1 Ferroptosis in cancer therapy.

Ferroptosis Mechanism

Ferroptosis is a new form of cell death characterized by iron-dependent accumulation of lipid peroxidation to lethal levels.16 It has been shown that in mammalian cells, ferroptosis is mainly regulated by iron homeostasis, lipid metabolism, and glutathione-dependent redox homeostasis (Figure 2).

Figure 2 Ferroptosis mechanisms and their associated inducers of ferroptosis.

Abbreviations: TF, Serum Transferrin; TFR1, The membrane protein Transferrin Receptor1; NCOA4, Nuclear receptor coactivator 4; STEAP3, Six-Transmembrane Epithelial Antigen of Prostate 3; DMT1, Divalent Metal Transporter 1; PE-PUFA, Phosphatidyl Ethanolamine-Polyunsaturated Fatty Acids; LOX, Lipoxygenase; Ferroportin1, FPN1, Ferritin; Membrane iron transport protein 1; PL-PUFA-OOH, Lipid hydroperoxide; ACSL4, Acyl-CoA Synthetase Long-Chain Family Member 4; SLC3A2, Recombinant Solute Carrier Family 3, Member 2; MDA, Malondialdehyde; 4HNE, 4-hydroxynonenal; POR, Cytochrome P450 oxidoreductase; FMN, Flavin mononucleotide; FAD, Flavin adenine dinucleotide; SLC7A11, Recombinant Solute Carrier Family 7, Member 11; GCL, Glutamate-cysteine ligase; GSS, Glutathione synthase; GSH, Glutathione; GPX4, Glutathione Peroxidase 4; GSSG, Oxidized glutathione; Glu, Glutamic acid; Nrf2, Nuclear factor E2-related factor 2; tumor suppressor, p53; Keap1, Recombinant Kelch-like ECH Associated Protein 1; ARE, Antioxidant response element; HO-1, Heme oxygenase-1; FSP1, Ferroptosis Inhibitory Protein 1; CoQ10, Coenzyme Q10; CoQ10H2, Panthenol; NADPH, Nicotinamide Adenine Dinucleotide Phosphate; Ipp, Isopentenyl pyrophosphate.

Iron Homeostasis

Iron is a trace element essential for maintaining the life of living organisms. It is involved in various metabolic pathways and exists in the human body mainly in Fe2+ and Fe3+.17 Two molecules of Fe3+ can bind to a transferrin (TRF) for transport in vivo and transport iron into the cell by binding to the transferrin receptor TFR1 on the cell surface to form the TF-Fe3+-TFR1 complex.18 Fe3+ is reduced to Fe2+ by the Six-Transmembrane Epithelial Antigen of Prostate 3 (STEAP3),19 which accumulates in cells to form the labile iron pool (LIP).20 SLC11A2 regulates this process. Fe2+ in LIP participates in the Fenton reaction to generate reactive oxygen species (ROS) substances represented by hydroxyl radicals (·OH).21 Accumulated ROS will undergo lipid peroxidation, resulting in loss of cellular function and cellular ferroptosis.22 Autophagic ferritin degradation in lysosomes mediated by nuclear receptor coactivator 4 (NCOA4) can induce ferroptosis by releasing free iron from ferritin.23 In iron metabolism, the Feature Pyramid Network (FPN) is the only protein that can translocate iron from the cell, thereby reducing the intracellular Fe2+ concentration.24

Lipid Metabolism

Lipid metabolism is strongly associated with ferroptosis.25 Lipid peroxidation, a process in which oxygen combines with lipids to form peroxyl radicals to produce lipid peroxides, is critical to the onset of ferroptosis.26 Polyunsaturated fatty acids (PUFAs) containing two or more double bonds are involved in the lipid oxidation process of ferroptosis.27 Arachidonic acid (AA) or adrenaline (AdA) was acylated to AA/AdA-CoA by Abstract Long-chain acyl-coenzyme A (CoA) synthase 4 (ACSL4) and then by Lysophosphatidylcholine acyltransferase 3 (LPCAT3) to PE-AA/AdA.28 PE-AA/AdA is involved in downstream ferroptosis’s non-enzymatic and enzymatic oxidation reactions.29 Non-enzymatic reactions refer to the reaction of ·OH produced by the Fenton reaction with lipids to form lipid peroxides, which eventually undergo lipid peroxidation, leading to ferroptosis.30 Enzymatic reactions include two kinds. One is that PE-PUFA is catalyzed by lipoxygenase (LOX) to form toxic products such as Malondialdehyde (MDA) and 4-Hydroxynonenal (4-HNE), which react with DNA bases, proteins, and other nucleophilic molecules to cause severe harmful effects.31 The other is Cytochrome P450 reductase (POR), which promotes lipid peroxidation and leads to ferroptosis.32 Thus, ACSL4, LOX, and LPCAT3 are compelling targets for the action of ferroptosis inducers.

Antioxidant Pathways

The intracellular antioxidant pathway resists the onset of ferroptosis by neutralizing lipid peroxides and is a critical ferroptosis defense system in the body.33 Inhibiting antioxidant pathways disrupts ferroptosis defenses, leading to the induction of ferroptosis. Described below are a few of the major pathways involved in the induction of ferroptosis.

Xc−-GSH-GPX4

The Xc−-GSH-GPX4 axis is a central regulatory pathway for ferroptosis.34 The Cystine/Glutamate Antiporter (System Xc−) is on the cytoplasmic membrane. It contains solute carrier family 3 member 2 Gene (SLC3A2) and solute carrier family 7 member 11 Gene (SLC7A11, xCT), which is responsible for the transfer of cysteine(Cys) into the cell and glutamate(Glu) out of the cell.35 Cysteine that is moved into the cell by system Xc− can synthesize glutathione (GSH), which has antioxidant properties and strong reducing properties, with glutamate and glycine catalyzed by Glutathione synthetase (GSS) and Glutamate-cysteine ligase (GCL).36 GPX4 converts GSH in cells to oxidized glutathione (GSSG).37 In this process, GSH acts as an electron donor to convert toxic lipid peroxides in the cell to non-toxic Lipid alcohol (PL-PUFA-OH).38 GSH and GPX4 are essential for maintaining intracellular redox balance and inhibiting the occurrence of ferroptosis. Inhibition of GSH synthesis through inhibition of system Xc− and direct inhibition of GPX4 are important mechanisms of action of ferroptosis inducers. Selenocysteine is one of the essential amino acids for the active group of GPX4.39 Thus, selenocysteine is an important target for a variety of GPX4 inhibitors.

FSP1-CoQ10-NAD (P) H Pathway

The FSP1-CoQ10-NAD(P)H pathway and the GSH-GPX4 pathway are two parallel lipid antioxidant pathways.40 CoQ10 (ubiquinone) is a lipophilic free radical trapping antioxidant that blocks the propagation of lipid peroxides and inhibits ferroptosis.41 Its reduced form of CoQ10H2 (panthenol) reduces oxidative stress and inhibits lipid ROS accumulation.42 Ferroptosis suppressor protein 1 (FSP1) reduces CoQ10 to CoQ10H2 in response to NADPH and catalyzes the regeneration of CoQ10 via NAD(P)H.43 The FSP1-CoQ10-NAD(P)H pathway exerts antioxidant effects independently of the system Xc−-GSH-GPX4 axis and inhibits ferroptosis. Inhibition of FSP1 blocks the antioxidant effects of the FSP1-CoQ10-NAD(P)H pathway, thereby inducing ferroptosis.40

Nrf2/ARE-GPX4 Pathway

Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in cellular antioxidant capacity. Nrf2 regulates the expression of antioxidant and pro-electrical stress genes. It also participates in other important pathways such as lipid metabolism, iron homeostasis and energy metabolism.44 Kelch-like ECH-associated protein 1 (Keap1) is an active inhibitor of Nrf2. Under normal conditions, Nrf2 binds to Keap1. Under oxidative stress, Nrf2 is detached from the Keap1 binding site. It rapidly translocates to the nucleus, interacting with the Antioxidant Response Element (ARE) in the promoter region of mRNAs encoding detoxification enzymes and cytoprotective genes to enhance the expression of downstream target genes.45 The downstream target genes of Nrf2 include genes related to iron metabolism, genes related to NADPH regeneration, and genes related to GSH metabolism.46 Activation of these target genes will effectively maintain the redox homeostasis of the cell. Nrf2 also binds to Heme Oxygenase-1(HO-1) and inhibits the occurrence of lipid peroxidation.47 Furthermore, activation of Nrf2 reduces cellular iron uptake, increases iron storage, and limits ROS production.48 This shows that Nrf2 plays an important protective role against ferroptosis. Inhibition of Nrf2 is also a very important mechanism of action for ferroptosis inducers.49

Mevalonate Pathway

The Mevalonate pathway (MVA pathway) includes the generation of isopentenyl pyrophosphate (IPP), squalene and CoQ10.50 Selenocysteine is one of the amino acids in the active center of GPX4, and embedding it in GPX4 requires a special transporter, selenocysteine tRNA.51 IPP promotes selenocysteine tRNA maturation and GPX4 synthesis. Also, IPP is a precursor of CoQ10 and can synthesize CoQ10 in the presence of acetyl-coenzyme A (Acetyl-CoA).52 Thus, the MVA pathway connects the system Xc−-GSH-GPX4 axis and the NADPH-FSP1-CoQ10 pathway.

p53 Gene

The p53 gene is a classical oncogene. As a transcription factor, p53 activates or represses the transcription of multiple downstream target genes to perform its function.53 The p53 can transcriptionally repress SLC7A11 expression at the transcriptional level, thereby promoting the occurrence of ferroptosis in cells.54 Also, p53 releases Arachidonate 12-lipoxygenase (ALOX12). Free ALOX12 oxidizes cell membrane phospholipids’ polyunsaturated fatty acid chains, leading to ferroptosis.55

Ferroptosis Inducers Traditional Small Molecular Compounds

With the increasing research on ferroptosis, more and more inducers targeting ferroptosis have been identified and developed. Traditional small molecular compounds are an essential component of the ferroptosis inducer field. The following section will summarise these ferroptosis inducers in terms of the pathways of action of these drugs (Table 1).

Table 1 Small Molecular Ferroptosis Inducers

Targeting Antioxidant Pathways Targeting System Xc−

The synthesis of glutathione (GSH) heavily relies on the cystine imported into the cell through system Xc−.130 Hence, system Xc− significantly influences the progression of ferroptosis. Conversely, the inhibitory function of system Xc− hinders cystine uptake, leading to decreased GSH production, cellular stress, and ultimately triggering ferroptosis.131

Erastin, a well-known inducer of ferroptosis, was first discovered in 2003 by Dolma et al.132 This quinazolinone derivative inhibits system Xc− by targeting SLC7A11.133 Moreover, erastin promotes oxidation and disruption of the mitochondrial permeability transition pore (mPTP) and acts through the Voltage-Dependent Anion Channel (VDAC), demonstrating anti-tumour effects.134 Erastin effectively triggers ferroptosis in various cell types, including human promyelocytic leukemia cells (HL60),135 endometriotic ectopic cyst cells (EESC),136 and renal tubular cells.137 Not only does erastin induce ferroptosis in cancer cells, but it also enhances the efficacy of traditional anticancer drugs like adriamycin,138 cisplatin,139 temozolomide,140 and cytarabine141 on specific cancer cell lines. However, Erastin’s limitations in stability, water solubility, and pharmacokinetic properties render it unsuitable for in vivo applications.142

The quinazolinone present in erastin’s structure is crucial for its cell-inhibitory effects. By modifying the structure of erastin, researchers have developed several drugs that outperform erastin. Wan Seok Yang et al incorporated a Piperazine Fragment into erastin’s aniline to create Piperazine erastin (PE).59 PE exhibits higher water solubility and metabolic stability compared to erastin (water solubility: 0.086 × 10−3M for erastin, 1.4 × 10−3M for PE). Imidazole ketone erastin (IKE) is an erastin analogue containing a carbonyl group, designed by Marie-Helene Larraufie et al.143 IKE induces ferroptosis and demonstrates anti-tumour effects in a diffuse large B cell lymphoma (DLBCL) xenograft model.144 IKE shows a superior inhibition rate and metabolic stability when compared to erastin, making it more suitable for in vivo assessments and offering promising application prospects.144

Yuying Fang et al utilized phenotypic screening to discover a novel ferroptosis inducer known as 2-(trifluoromethyl)benzimidazole derivatives FA-S.61 They further developed 30 derivatives based on this scaffold, with FA16, containing the 4-SO2NMe2 group, demonstrating the highest lethality. By inhibiting the Xc− transporter, FA16 effectively triggers ferroptosis in various human cancer cell lines.61 The IC50 value of FA16 in HT1080 cells was measured at 1.26 μM. Moreover, in the HepG2 xenograft model, FA16 displayed significant inhibition of tumor growth. Both in vivo and in vitro studies have confirmed the favorable safety profile of FA16.61

Lepadins, natural alkaloids derived from the marine didemnum family, exhibit diverse biological activities including anti-cancer and anti-malarial properties.145 Wenjun Wang et al introduced a novel synthetic approach to get seven compounds within the Lepadins series. Notably, lepadins E and H demonstrated notable cytotoxicity in cellular assays, enhancing p53 expression, elevating ROS and lipid peroxidation levels, reducing SLC7A11 and GPX4 expression, up-regulating ACSL4, and triggering ferroptosis through the classical p53-SLC7A11-GPX4 pathway.62 In animal studies, Lepadin H effectively suppressed melanoma growth. Nevertheless, Lepadins E and H exhibited only modest selectivity towards cancer cells over normal cells, necessitating further investigation into their safety profile.62

Solasonine (SS) is a natural alkaloid-like compound extracted from potato tubers with anti-cancer and anti-arthritic effects.146 Research has demonstrated that SS exhibits toxicity towards various cancer cell lines, including hepatocellular carcinoma,147 lung cancer,148 and gastric cancer cells.149 The induction of ferroptosis in lung adenocarcinoma by SS is linked to redox imbalance and mitochondrial dysfunction.150 In pancreatic cancer cells, SS triggers ferroptosis through the TFAP2A/OTUB1 SLC7A11 axis,71 while in liver cancer cells, it disrupts the GSH-GPX4 redox system to induce ferroptosis.151

Curcumin, a polyphenolic compound derived from turmeric rhizomes, possesses chologogic, antimicrobial, and anti-cancer properties.152 Studies have shown that curcumin induces ferroptosis in various tumors such as non-small cell lung cancer,73 breast cancer,74 and glioblastoma,153 each with distinct mechanisms. For instance, curcumin inhibits breast cancer by up-regulating SLC1A5 expression, a crucial glutamine transporter.74 In follicular thyroid carcinoma, curcumin induces ferroptosis by increasing Heme Oxygenase-1 (HO-1) expression.154 The induction of ferroptosis in non-small cell lung cancer and hepatic stellate cells involves the activation of autophagy.155

Sulfasalazine (SAS) is an anti-inflammatory agent commonly used to treat chronic inflammatory diseases.156 It is a potent inhibitor of system Xc− that characteristically inhibits cystine transport mediated by SLC7A11.54 SAS can effectively induce ferroptosis in tumor cells such as Multiple myeloma (MM) cells,157 Esophageal cancer (EC) cells63 and TNBC cells.158 Sorafenib is a protein kinase inhibitor approved for the treatment of hepatocellular carcinoma and advanced renal cell carcinoma.159 It attenuates hepatic fibrosis and liver injury by inhibiting systemic Xc− induced ferroptosis in hepatic stellate cells.160 Sorafenib treatment was accompanied by a significant increase in Cyclooxygenase-2 (COX2) expression and a decrease in SLC7A11 and GPX4 proteins. Yun-Jeong Kim et al designed and synthesized JB3, a derivative compound of sorafenib, which showed significant tumor growth inhibition in both in vivo and in vitro experiments.128 However, it has also been suggested that sorafenib fails to trigger ferroptosis in a wide range of cancer cell lines.161 Its cytotoxic effects in various cell lines, including HT1080, HEK293T and B16F10 cells, could not be prevented by ferroptosis inhibitors. The ability and mechanism of action of sorafenib as an ferroptosis inducer remains controversial. Marketed drugs such as the PARP inhibitor olaparib,162 the anti-malarial medicine flubendazole,67 and the anti-leukemic drug 6-Thioguanine (6-TG),68 which act on a variety of diseases, have also been found to Anti-tumour effect by inhibiting system Xc−-induced ferroptosis in cells. The classic Antidiabetic Drug metformin has also been found to inhibit breast cancer by inducing ferroptosis through down-regulation of SLC7A11 expression.69

Targeting GSH-GPX4

GPX4 is a central regulator of ferroptosis, and inhibition of GPX4 is an important mechanism of action for ferroptosis inducers.

RAS-selective lethal 3 (RSL3) is a Tetrahydro-β-carboline (THBC) identified by Wan Seok Yang et al in 2008.163 As a well-known inducer of ferroptosis, RSL3 effectively hinders the growth and proliferation of various cancer cells, such as those found in lung, breast, and liver cancers. By directly binding to selenocysteine residues in the GPX4 catalytic site, RSL3 deactivates them, leading to an increase in ROS and cellular LIP levels. This results in the accumulation of intracellular lipid peroxides and triggers ferroptosis.164 It has been observed that while RSL3 alone does not induce ferroptosis in glioblastoma, its combination with NF-κB pathway activation does.75 However, the clinical use of RSL3 is hindered by side effects and drug resistance, necessitating further research and improvement for its application.165

Congjun Xu et al synthesized a series of novel and efficient GPX4 inhibitors using RSL3 as lead-compound and scaffold hopping strategy.77 By determining the compounds’ IC50 values and GPX4 inhibition in various cells, compound 26a containing Oxazole Fragment was identified as the most superior GPX4 inhibitor. Compound 26a inhibited GPX4 by 71.7% at 1.0 μM with an IC50 value of 0.15 μM in HT1080 cells.77 It significantly inhibited tumor growth in a 4T1 xenograft model without significant toxicity. 26a ameliorates the selectivity and cytotoxicity problems of conventional chloroacetamide-based GPX4 inhibitors.77

In addition, John T. Randolph et al designed and synthesized ferroptosis inducer using RSL3 as a lead compound 24.78 Compound 24 has similar cell-killing activity (IC50 = 0.117 μM) and higher plasma stability (t1/2 >5 h) in mouse plasma compared to RSL3, but it has limited efficacy as a single-agent tumor-targeting therapy.78

Michel Weïwer et al identified two novel molecular probes ML162 and ML210 by high throughput screening.79 Both ML162 and ML210 are covalent inhibitors of GPX4, inactivating GPX4 selenocysteine residues by covalently binding them. ML162 is similar to RSL3 and also has the chloroacetamido group. However, it is now proposed that RSL3 and ML162 are not direct inhibitors of GPX4 but Thioredoxin reductase 1 (TXNRD1).166 TXNRD1 is another cytoplasmic selenoprotein that regulates intracellular redox and is a newly proposed key regulator and drug target for ferroptosis.167 ML210 is a compound containing nitroisoxazoles. ML210 has an intact cell-dependent inability to interact with purified GPX4 protein. It must be activated in intact cells to produce the critical metabolite JKE-1674 and undergo additional cellular activation and dehydration to generate an electrophile JKE-1777 that binds to GPX4.168 GPX4 inhibitors acting via nitrile oxide exhibited significant selectivity for GPX4 and reduced off-target effect compared to chloroacetamide-based GPX4 inhibitors.168

Conventional chloroacetamide GPX4 inhibitors suffer from off-target effect, poor stability and selectivity, limiting their use in in vivo therapy.169 In order to optimize these inhibitors, Tingting Chen et al performed structural optimization of RSL3 and ML162 based on a molecular docking model that retained the chloroacetone fragment and introduced N-benzylaniline and diphenylamine skeleton and successfully synthesized a series of novel GPX4 covalent inhibitors.80 Among them, compound C18 covalently binds to Sec46 of GPX4 and inhibits the function of GPX4, leading to ferroptosis in TNBC cells. Compound C18 showed a higher inhibition rate, better GPX4 inhibitory activity and in vivo antitumor pharmacokinetic properties compared to RSL3 and ML162. At a concentration of 1 μM, C18 almost completely inhibited the activity of TNBC cell lines and showed high selective inhibition of these cell lines without significant organ toxicity.80

Furong Ma et al used pharmacophore hybridization and bioisosterism strategies to construct a series of ML162-quinone conjugates.129 Among them, GIC-20 was identified as the most active compound. The IC50 of GIC-20 in HT-1080 cells is 1.6 μM. GIC-20 not only induces ferroptosis through proteasome-mediated GPX4 degradation but also induces apoptosis by up-regulating the level of the apoptotic protein Bax and down-regulating the level of the antiapoptotic protein Bcl −2.129 Meanwhile, GIC-20 showed no significant toxicity compared with ML162.

Ferroptosis inducers targeting GPX4, also identified by screening, include FIN56,81 N6F11170 and Metamizole sodium.83 FIN56 not only promotes GPX4 degradation via Acetyl-CoA carboxylases (ACCs), but also degrades GPX4 by binding to and activating squalene synthase, an enzyme involved in cholesterol synthesis, thereby inducing ferroptosis.171 FIN56 can effectively exert anti-tumour effects. N6F11 binds to a specific structural domain of the ubiquitin E3 ligase TRIM25, predominantly expressed in tumor cells, triggering ubiquitination and degradation of GPX4.82 Thus, in contrast to conventional GPX4 inhibitors, N6F11 can selectively induce GPX4 protein degradation in tumor cells without causing damage to immune cells.

Unlike traditional GPX4 inhibitors targeting the active site selenocysteine, Hengrui Liu et al identified an unexpected variant site C66 on GPX4. They screened the lead compound LOC1886, which covalently binds this allosteric site to inhibit GPX4.84 However, LOC1886 showed lower efficacy than RSL3 and ML162 in inducing cellular ferroptosis.

CH004 is a biologically active inhibitor of cystathionine β-synthase (CBS) with in vitro and in vivo effects designed and synthesized by Li Wang et al.85 CH004 dose-dependently inhibits CBS activity in cells or in vivo, inhibiting cysteine and hydrogen sulfide production. CH004 can effectively inhibit tumor growth in vivo and in vitro with an IC50 value of 1 μM.85 A compound that also indirectly inhibits GPX4 is Buthionine sulfoximine (BSO). BSO is a fast-acting and irreversible inhibitor of glutamate cysteine ligase (GCL), which inhibits glutathione synthesis, indirectly inactivates GPX4, induces lipid peroxidation, and thus inhibits cell viability, and has been shown to have an inhibitory effect on breast cancer, melanoma, and Others.172 However, its therapeutic effect as an ferroptosis inducer is only seen in tumors with low levels of GSH expression. Simvastatin inhibits hepatic stellate cell (HSC) activation and induces ferroptosis in HSC by inhibiting the MVA pathway to downregulate GPX4 expression.87

Orlistat is an anti-obesity drug approved by the US Food and Drug Administration (FDA), and fatty acid synthase (FASN) is the main lipomatoses it inhibits.88 Studies have shown that Orlistat induces ferroptosis-like cell death of lung cancer cells.89 Orlistat reduces GPX4 expression, upregulates phosphorylated MAPK/ERK, and induces lipid peroxidation in lung cancer cells. Orlistat also promotes lipid peroxidation and ferroptosis in pancreatic neuroendocrine tumors (pNETs) cells by inhibiting the expression of xCT and GPX4 and increasing the expression of CD71.173

Tubastatin A, an inhibitor of Histone deacetylase-6 (HDAC6), significantly induces ferroptosis in ferroptosis-sensitive cell lines, and this process was not dependent on its inhibition of HDAC6.174 Radiotherapy not only induces ferroptosis by promoting ROS generation and ACSL4 expression in cancer cells, but also increases ferroptosis-resistance and radio-resistance of cancer cells by activating Nrf2-mediated GPX4 transcription and inhibiting lysosome-mediated GPX4 degradation.90 Tubastatin A can overcome ferroptosis-resistance and radio-resistance in cancer cells by directly binding to GPX4 and inhibiting GPX4 enzyme activity.174 In the xenograft model, it significantly promotes radiotherapy-induced lipid peroxidation and effectively improves tumor suppression. However, Tubastatin A is a hydroxamic acid and may cause some side effects in subsequent clinical trials.174 Therefore, optimization of the structure of Tubastatin A is essential.

Gliotoxin91 and Timosaponin AIII92 are two natural products that induce ferroptosis by targeting GPX4. In addition to targeting GPX4, Gliotoxin was found to act on the protein SLC7A11 in H1975 cells.91 Timosaponin AIII promotes ferroptosis in NSCLC by targeting and promoting heat shock protein 90 (HSP90)-mediated GPX4 ubiquitination and degradation.92 Compound 31 is an indirubin derivative synthesized by Jiang-Min Zhu et al by combining the core of the natural product indirubin with the active fragment of a GPX4 covalent inhibitor via a linker.93 The IC50 of compound 31 in HCT-116 cells was 0.49 μM. It significantly reduced GPX4 levels in HCT-116 cells by promoting GPX4 ubiquitination.

Sulforaphane (SFN) is a naturally occurring isothiocyanate found in various vegetables that has anti-cancer and anti-inflammatory properties.175 Recently, Giulia Greco et al showed that in leukemia cells SFN triggers different patterns of cell death in a dose-dependent manner.94 At 25 µM, SFN induced caspase-dependent apoptosis, and at 50 µM, ferroptosis was induced through GSH depletion, decreased GPX4 expression and lipid peroxidation. The anti-leukemic activity of SFN can be mediated by inducing both ferroptosis and apoptosis.94

Targeting FSP1

The FSP1-CoQ10-NAD(P)H pathway and the xCT-GSH-GPX4 pathway are two parallel lipid antioxidant pathways.176 The two pathways act synergistically to inhibit lipid peroxidation and ferroptosis. Inhibition of FSP1 would effectively inhibit the antioxidant effects of the FSP1-CoQ10-NAD(P)H pathway, thereby inducing the onset of ferroptosis.177

Sebastian Doll et al conducted a screening of around 10,000 compounds, resulting in the discovery of a potent FSP1 inhibitor named iFSP1, which is the first targeted FSP1 inhibitor described in the literature.178 iFSP1 induces ferroptosis to inhibit cancer cell growth in vitro by inhibiting GPX4. The IC50 value of iFSP1 is 103nM. iFSP1 is specific for hFSP1, and residue F360 within hFSP1 is closely related to the binding of iFSP1 and its target. iFSP1 can inhibit the growth of hepatocellular carcinoma (HCC) by inducing ferroptosis and potential immunomodulatory effects.95 Up-regulated chemokines after iFSP1 treatment promote infiltration of antigen-presenting cells, macrophages and cytotoxic T cells into the tumor.

Toshitaka Nakamura et al predicted the physicochemical properties and drug similarities of about 10,000 small molecule drugs and finally identified 3-phenylquinazolinones as a class of potent inhibitors of FSP1.96 Of these, icFSP1 is the most representative. icFSP1 is a specific inhibitor of hFSP1, which triggers the Phase separation of FSP1 by inducing the formation of FSP1 condensate in tumor cells to induce ferroptosis in cancer cells.96 In contrast to iFSP1, icFSP1 did not show off-target effects even at higher concentrations.

Joseph M. Hendricks et al identified several structurally distinct FSP1 inhibitors through chemical screens. Among them, FSEN1, a non-competitive inhibitor of FSP1, showed the highest potency in inducing cell death in NCI-H460 GPX4KO cells (EC50 = 69.363 nM).97 However, in most cells, FSEN1 could not induce ferroptosis by inhibiting FSP1 alone. In vitro experiments showed that FSEN1 synergizes with dihydroartemisinin, an endoperoxide-containing ferroptosis inducer, to induce ferroptosis in tumor cells.97

iFSP1, icFSP1, and FSEN1 specifically target hFSP1 and fail to be tested in vivo in tumor transplantation models. Whether they inhibit FSP1 in vivo and whether they have an effect on tumor growth remains to be investigated. Toshitaka Nakamura et al identified the first non-species-dependent FSP1 inhibitor, versatile inhibitor of FSP1 (viFSP1), which targets the NAD(P)H binding pocket.98 Treatment with viFSP1 in Pfa1 cells expressing mouse FSP1 or hFSP1 led to significant lipid peroxidation and ferroptosis.

Compound NPD4928, screened from a chemical library by Hiromasa Yoshioka et al, also targets binding to FSP1 and inhibits its activity.99 The combination of NPD4928 and GPX4 inhibitors induced ferroptosis; however, using FSP1 alone did not induce ferroptosis and was not sensitive in some cells. In addition to these drugs, curcumin179 and sorafenib180 have been found to induce ferroptosis in cells via the FSP1 pathway.

The study reveals that most FSP1 inhibitors alone are unable to induce ferroptosis in cells, or can only do so in cells with low GPX4 expression.181 Instead, these inhibitors primarily act as enhancers of ferroptosis and are often combined with other ferroptosis inducers to enhance their effects.182

Targeting Nrf2

Nrf2 is involved in the regulation of oxidative homeostasis in vivo by acting on downstream target genes.183 Inhibition of Nrf2, and thus its antioxidant effects, is also an important pathway of action for ferroptosis inducers.184

S-3’-hydroxy-7’, 2’, 4’-trimethoxyisoxane (ShtIX) is a novel isoflavone compound derived from the heartwood of Dalbergia latifolia.100 ShtIX has demonstrated broad-spectrum anticancer activity, with particularly high cytotoxicity against NSCLC cells. Research has shown that ShtIX induces ferroptosis in NSCLC cells by targeting the Nrf2/HO-1 signaling pathway, with an IC50 of 7.9 µM in A549 cells. Importantly, ShtIX selectively eliminates non-small cell lung cancer cells.100

Trigonelline, the primary alkaloidal component of the legume herbaceous Trigonella foenum-graecum, exhibits anti-HSV-1, antibacterial, and antifungal activities.185 Acting as an Nrf2 inhibitor, trigonelline reduces cellular resistance to ferroptosis and shows potential for combination therapy with other ferroptosis inducers.101 In xenograft models, trigonelline enhances the anticancer effects of erastin, sorafenib, and RSL3, and increases the chemosensitivity of Etoposide in pancreatic cancer.186

Baicalin, a flavonoid extracted from the dried root of Lamiaceae family plants Scutellaria baicalensis Georgi, possesses antibacterial, antiallergic, antitumor, and other pharmacological properties.187 Research indicates that baicalin can induce ferroptosis in bladder cancer cells by activating Ferritin Heavy Chain 1 (FTH1).104 In osteosarcoma cells, baicalein interacts with Nrf2, affecting their stability by promoting ubiquitination and degradation, leading to the inhibition of GPX4 and xCT expression, ultimately inducing ferroptosis in osteosarcoma cells.188

Matrine, an alkaloid derived from the roots of Sophora flavescentis Radix (Kushen), exhibits notable antitumor properties.105 Research has demonstrated that matrine can trigger ferroptosis in various types of tumor cells, including cervical cancer cells,189 rectal cancer cells,190 and hepatocytes.191 The specific mechanisms through which matrine induces ferroptosis in different cell types vary. For instance, in cervical cancer, matrine hinders cervical cancer progression by activating the Piezo1-Ca2+ signaling pathway to induce ferroptosis.189 Additionally, matrine induces ferroptosis in hepatocytes by suppressing the Nrf2/GPX4 antioxidant system.191

Acetaminophen (APAP) is a commonly used antipyretic and analgesic medication that can lead to liver damage in a dose-dependent manner when taken in excess, potentially causing acute liver failure in severe cases.192 Research indicates that the primary mechanism underlying APAP-induced liver damage is ferroptosis, although the exact process by which it triggers ferroptosis in liver cells remains unclear.193 Studies have revealed that NSCLC cell lines exhibit reduced sensitivity to erastin-induced ferroptosis, but APAP can enhance this sensitivity by modulating the Nrf2/HO-1 signaling pathway.102 Similarly, Sunitinib (SUN), a multi-targeted tyrosine kinase inhibitor known for its anti-angiogenic and anti-tumor properties, is associated with cardiotoxic effects that limit its clinical utility.103 It has been found that Nrf2-dependent ferroptosis plays a critical role in SUN-induced cardiotoxicity.194

Targeting Lipid Metabolism

Lipid peroxidation is central to the development of ferroptosis, and regulation of lipid metabolism is also a critical action pathway for ferroptosis inducers.

Shuai Hua et al developed a series of quinazolinediones (QD)-based ROS inducers and screened the optimal compound, QD325, after testing it in pancreatic cancer preclinical models.114 They synthesized a set of QD derivatives by making structural modifications to QD325. QD394 was the optimal compound. The transcriptome profile of QD394 closely resembles that of napabucasin, a cancer stem cell inhibitor. Both small molecules inhibited STAT3 phosphorylation, increased cellular ROS, and decreased the GSH/GSSG ratio.114 The IC50 value of QD394 in PANC-1 cells was 0.34 μM. It was optimized to give the derivative QD394-Me, which showed better plasma stability and reduced toxicity in mice compared to QD394. The IC50 value of QD394-Me in PANC-1 cells was 0.38 μM. QD394-Me, as ROS-inducing drugs, exhibited potent antitumor effects on human pancreatic cancer cells.114

Imetelstat, a telomerase inhibitor, competitively inhibits telomerase activity by targeting the telomerase RNA template and is primarily used in the clinical treatment of myelofibrosis and myelodysplastic syndromes.115 Claudia Bruedigam et al discovered that imetelstat, a potent inducer of ferroptosis, effectively reduced the burden of acute myeloid leukemia (AML) and delayed relapse after oxidative stress-induced therapy.195 Imetelstat increases the synthesis and subsequent oxidation of PUFA phospholipids, and its induced lipophagy triggers ferroptosis in AML. Imetelstat causes cell death by involving two regulators of fatty acid metabolism (FADS2 and ACSL4), leading to excessive lipid reactive oxygen species formation and ferroptosis.196

Tingting Liang et al designed and synthesized a series of novel urea derivatives, among which the compound 10p exhibited the most effective antiproliferative activity against HT-29 cells.127 10p could induce both ferroptosis and autophagy in cells. It was shown that 10p-induced cell cycle arrest, ferroptosis, and autophagy may be related to the accumulation of ROS. 10p is a promising lead compound that can be used for further optimization.

Disulfiram (DSF), a specific inhibitor of aldehyde dehydrogenase type I (ALDH1), is commonly used in the treatment of alcoholism due to its acute sensitization to alcohol.197 DSF has been found to possess antitumor properties in various tumor types, with induction of ferroptosis being one of its mechanisms.197 DSF triggers lysosomal membrane permeabilization in a ROS-dependent manner, a key mechanism for inducing cell death.116 Additionally, DSF enhances cell sensitivity to chemotherapy and radiotherapy.198 Furthermore, DSF exhibits copper-dependent anticancer activity, and when combined with copper, its ability to induce tumor cell death is significantly enhanced. Studies have shown that DSF/Cu effectively induces ferroptosis in TNBC cells.199

Quercetin is a flavonoid that has shown anticancer activity in various cancers, including lung, ovarian, and prostate cancers.200 Induction of ferroptosis is one of its pathways of action against cancer. Quercetin increases ROS and transferrin receptor production, decreases mitochondrial membrane potential, GPX4 and SLC7A11 levels, and inhibits NF-κB and Nrf2.117

Darapladib is an orally active, selective and reversible inhibitor of phospholipase A2s (PLA2s).201 Lp-PLA2 is located in the cell membrane and cytoplasm, controls intracellular phospholipid metabolism and contributes to ferroptosis resistance.118 In the xenograft model, darapladib, combined with the GPX4 inhibitor PACMA31, sensitized cells to ferroptosis and showed antitumor activity that inhibited tumor growth.118

Auranofin (AUR), a therapeutic agent for rheumatoid arthritis, is a novel modulator of hepcidin expression.119 AUR can reduce iron overload by upregulating Hepcidin expression through activation of the NF-κB/IL-6/JAK-STAT signaling pathway, suggesting that AUR is a potential new drug for the treatment of hereditary hemochromatosis and iron overload-associated diseases triggered by hepcidin deficiency. However, when given at high doses (25 mg/kg bw), AUR triggered hepatic ferroptosis and 100% mortality by inhibiting TXNRD activity and increasing lipid peroxidation.119

Targeting Iron Metabolism

Abnormal iron metabolism plays a significant role in ferroptosis, with iron metabolic pathways being a key target for regulation of this process.

Hinokitiol, a natural compound derived from tropolone, acts as an iron chelator with various beneficial properties such as anti-inflammatory, antioxidant, antibacterial, and antifungal effects.202 Research by Hongting Zhao et al revealed that the Hinokitiol-iron complex Fe(hino)3, formed by combining hinokitiol and iron in a 3:1 ratio, serves as an inducer of ferroptosis.120 This redox-active complex triggers lipid peroxidation and decreases GSH-GPX4 production through the generation of ·OH via the Fenton reaction upon cellular entry, ultimately leading to ferroptosis induction in TNBC. Notably, serum biochemical indices of Fe(hino)3-treated tumor-bearing mice remained within normal ranges, suggesting a favorable safety profile of Fe(hino)3 in vivo.120

Steroidal saponin SSPH I, extracted from plantain herb, has been identified as an anti-hepatocellular carcinoma compound.203 Research has demonstrated that SSPH I exhibits significant antiproliferative and antimigratory effects on HepG2 cells. In these cells, SSPH I upregulates the expression of SLC7A5, a negative regulator of ferroptosis, while also increasing the expression of TFR and FPN proteins, which are positive feedback factors of ferroptosis. This leads to the accumulation of Fe2+ and subsequent iron overload, ultimately inducing ferroptosis.121 Additionally, SSPH I induces a notable G2/M blockade in HepG2 cells.121

Polyphyllin B (PB) is a dioscin isolated from Paris formosana with antitumor activity and immunomodulatory effects.122 Can Hu et al found that PB could effectively inhibit the proliferation of GC cells by inducing ferroptosis and apoptosis, and could prevent the migration, invasion and cell cycle of tumor cells in vitro. PB can regulate iron metabolism by promoting iron ion transport and ferritin autophagy, directly binding to GPX4 and reducing GPX4 expression to ultimately induce ferroptosis.122 Its anti-GC function may also be related to its regulation of the MAPK signaling pathway, PI3K-AKT signaling pathway, and TNF signaling pathway.

Ochratoxin A (OTA) is one of the fungal toxins, a toxic metabolite produced by toxin-producing strains of Aspergillus ochraceus and penicillium, which is severely nephrotoxic.204 It has been shown that ferroptosis is a potential mechanism by which OTA causes renal injury OTA disrupts renal cell iron homeostasis and induces ferroptosis by up-regulating the expression levels of the iron input genes TFR1 and FTH, down-regulating the expression level of FPN, and significantly increasing its negative regulator hepcidin.123

Juglone is a natural product discovered from Juglans regia that has a variety of pharmacological properties, including antibacterial, antitumor, antiviral, and anti-inflammatory properties.205 Studies have shown that induction of ferroptosis is one of the mechanisms by which juglone is anti-tumor. The IC50 values of juglone in A549 cells and endometrial cancer cells were 18.5 µM and 20.81 µM. The mechanism of juglone-induced ferroptosis in A549 cells mainly involves an increase in iron accumulation and a decrease in GSH and MDA.125 In endometrial cancer, juglone activates HMOX1, which induces ferroptosis. Similar to juglone, strigolactones Bufotalin (BT) isolated from Venenum Bufonis induced ferroptosis in A549 non-small cell lung cancer cells by accelerating the degradation of GPX4 and increasing the intracellular Fe2+ level.206 The IC50 value for BT in A549 cells was 4.21 μM.

Withaferin A (WA) is a natural product isolated from withania that targets vimentin and has anti-inflammatory and anti-tumor properties.207 By inducing ferroptosis, WA inhibited cell viability, invasive ability, vasculogenic mimicry (VM) formation, and VE-cadherin promoter levels in HepG2 and SNU449 cells, thereby attenuating hepatocellular carcinoma metastatic potential and sorafenib resistance.126 WA has now been found to have multiple pathways for inducing ferroptosis. On the one hand, WA reduces the protein level and activity of GPX4. On the other hand, WA induces a novel atypical ferroptosis pathway by directly targeting Kelch-like ECH-Associated Protein 1 (Keap1), which increases unstable Fe2+ upon HMOX1 overactivation.126 WA also elevates Keap1 expression, attenuating Nrf2 signaling activation-mediated Epithelial-Mesenchymal Transition (EMT) and xCT expression.

The environmental pollutant benzo-a-pyrene (BaP) is a carcinogenic Polycyclic aromatic hydrocarbon, and BPDE (benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide) is the end product of the breakdown of BAP in the human body.124 BPDE is electrophilic and can covalently bind to the exocyclic amine terminus of guanine (G), a nucleophilic site of DNA, to form a BPDE-DNA complex. This complex causes DNA base mutations, thereby activating oncogenes and leading to malignant transformation of cells, ultimately resulting in tumorigenesis.124 Studies have demonstrated that BPDE induces ferroptosis in human trophoblast cells by upregulating iron metabolism, promoting GPX4 proteasome degradation, and increasing intracellular free Fe2+ and excess ROS, leading to lipid peroxidation.124

Dihydroartemisinin (DHA) in combination with cisplatin synergistically induces ferroptosis in pancreatic ductal adenocarcinoma cells by modulating iron metabolism.208 The combination of DHA and DDP significantly increased the expression of TFR and the accumulation of unstable free iron in pancreatic ductal carcinoma cells, impairing mitochondrial homeostasis. Co-treatment of DHA with DDP also synergistically reduced GPX4 expression. Additionally, the combined treatment of lapatinib (LAP) and doxorubicin (Dox) resulted in increased Fe2+ content, suppression of GPX4 expression, and elevation of ASCL4 levels in cardiomyocytes.209 These synergistic effects of LAP lead to pro-apoptotic, pro-mitochondrial dysfunction, pro-oxidative, and ferroptosis effects, resulting in cytotoxicity. Finally, the combination of lapatinib and the lysosomal pro-solubility drug siramesine induces ferroptosis by decreasing ferritin expression and increasing transferrin expression, ultimately leading to increased iron and ROS levels.111

Others

While research on ferroptosis and related drugs is progressing, there are still some ferroptosis inducers with unclear mechanisms of induction. For instance, the natural product ginkgolic acid (GA) inhibits SUMO1-activating enzyme subunit 1 (SAE1), a potential anti-fibrotic target protein.210 In both in vivo and in vitro experiments, GA was found to induce hepatic stellate cell ferroptosis by targeting SAE1, leading to an anti-hepatic fibrosis effect in a rat model of hepatic fibrosis.210 However, the relationship between SAE1 and ferroptosis remains unclear.210 On the other hand, small molecules EC330 and EC359 target the Transmembrane Receptor (LIFR) of the Leukemia inhibitory factor (LIF).211 By binding to LIFR at specific sites within the ligand-receptor complex, EC330/EC359 inhibit LIFR-induced activation of the STAT3 and AKT pathways, ultimately leading to cell death. Treatment with EC330/EC359 results in GPX4 inactivation and GSH depletion, potentially preventing cells from eliminating toxic lipid peroxides and triggering ferroptosis.212 However, the specific targets of EC330/EC359-induced ferroptosis require further investigation.

Statins are widely used, but atropostatin(Atv) administration leads to oxidative stress and cellular damage.213 Indicators of ferroptosis, such as iron overload, ROS accumulation, and lipid peroxidation, were observed in Atv-treated muscle cells, especially in the mitochondria of the cells.214 One study reported that simvastatin causes ferroptosis in cancer cells by inhibiting HMGCR expression.215 However, it has been shown that Atv can attenuate myocardial dysfunction and remodeling after isoproterenol stimulation through inhibition of ferroptosis, as well as attenuate de-ironing-dependent myocardial injury and inflammation after coronary microembolization through the Hif1a/Ptgs2 pathway.216 The specific role and mechanisms of Atv in ferroptosis continue to be studied.

JQ1, a small molecule inhibitor targeting the BET family protein BRD4, has been shown to inhibit the proliferation of cancer cells.217 Studies have shown that ferroptosis is one of the mechanisms by which JQ1 exerts its anticancer effects in vivo. JQ1 enhances ferroptosis by increasing ferritin autophagy or suppressing BRD4 regulation of ferroptosis-related genes.218 Expression of GPX4, SLC7A11, and SLC3A2 was downregulated under JQ1 treatment and BRD4 knockdown. However, the specific mechanism by which BRD4 regulates the expression of GPX4, SLC7A11 and SLC3A2 is unknown. JQ1 can induce ferroptosis in senescent cells by decreasing the expression of anti-ferroptosis genes in senescent cells,219 thereby selectively eliminating senescent cells. However, JQ1-induced ferroptosis occurred in both cancer and normal cells, and therefore, its safety for therapeutic application as an ferroptosis inducer requires further consideration.

Artesunate is a water-soluble hemisuccinate derivative of artemisinin used to treat malaria, improve inflammation, protect nerves, and treat tumors.220 In 2015, Edna Ooko et al noted that artemisinin and its derivatives induce ferroptosis in tumor cells.221 Co-action of artesunate and sorafenib induces ferroptosis in hepatocellular carcinoma cells; the former increases lysosomal activity and lipid peroxidation, and synergistically with the latter induces histone B/L activation and ferritin degradation.113 In addition, artesunate reduced GSH levels in head and neck cancer cells and ovarian cancer cells.222 However, it has also been shown that artesunate may activate the NRF2 antioxidant response element pathway, leading to resistance to ferroptosis.223

Proteolysis-Targeting Chimeras

Proteolysis-targeting chimeras (PROTACs) is a drug development technology that leverages the Ubiquitin-Proteasome System (UPS) to degrade target proteins.224 A typical PROTACs consists of three key components: E3 ubiquitin ligase ligands, target protein-ligand, and a specially designed “linker” structure that connects the two bioactive ligands.225 The application of PROTACs technology in modifying traditional ferroptosis drugs to enhance their targeting and antitumor properties represents a promising avenue for developing ferroptosis inducers (