Regulatory Mechanism

In recent years, ferroptosis, a novel form of cell death, has attracted widespread attention. Unlike traditional forms of cell death such as apoptosis and necrosis, ferroptosis is primarily driven by lipid peroxidation and iron metabolism abnormalities. The following is a detailed introduction to the regulatory mechanisms of ferroptosis, aimed at providing scientific support for related product promotion.

1. Basic Concept of Ferroptosis

Ferroptosis is a form of cell death dependent on iron and lipid peroxidation. In 2003, the Stockwell team first identified ferroptosis as a new form of cell death. Research indicates that iron plays a central role in this process by promoting lipid peroxidation reactions, ultimately leading to cell death. This process is distinctly different from classical forms of cell death such as apoptosis.

2. Classical Ferroptosis Regulatory Mechanisms

The classical regulatory mechanisms of ferroptosis involve the cystine/glutamate antiporter (system Xc-), glutathione (GSH), and glutathione peroxidase 4 (GPX4) axis.

• Cystine Uptake: Cystine enters the cell via the system Xc-transporter and is then converted to cysteine in the glutathione reduction pathway.
• Glutathione Generation: Cysteine is further converted to glutathione (GSH), which acts as a potent reductant. GSH, through the catalytic action of GPX4, reduces phospholipid hydroperoxides (PLOOHs) to phospholipid alcohols (PLOHs), thereby protecting the cell membrane structure.
• GSH Regeneration: Glutathione reductase (GSR) utilizes electrons provided by NADPH to reduce oxidized glutathione (GSSG) back to GSH.

In this process, the role of GPX4 is crucial. GPX4 is the primary enzyme for neutralizing PLOOHs. Its functional loss leads to the accumulation of PLOOHs, resulting in cell membrane damage and ferroptosis. Drugs like erastin and RSL3 can induce ferroptosis by inhibiting GPX4 through different mechanisms.

3. Phospholipid Peroxidation

Phospholipid peroxidation is a hallmark feature of ferroptosis. The initiation of lipid peroxidation reactions requires the removal of bis-allylic hydrogen atoms from polyunsaturated fatty acids (PUFAs) on phospholipids, forming free radicals. These free radicals react with molecular oxygen to generate peroxy radicals. If not promptly reduced, this can lead to cell membrane damage and cell death. ACSL4 and LPCAT3 are important enzymes; ACSL4 catalyzes the binding of PUFAs to coenzyme A, while LPCAT3 re-esterifies these products into phospholipids, thereby increasing the content of long-chain PUFAs in the cell membrane.

4. Cell Metabolism and Ferroptosis

The impact of cell metabolism on ferroptosis is gaining increasing attention. Studies have found that metabolic states can significantly affect the occurrence of ferroptosis. For example, glutamine metabolism is closely related to ferroptosis. Iron autophagy during the autophagic process can increase intracellular iron levels, promoting ferroptosis. Additionally, mitochondrial metabolism, glucose metabolism, and lipid synthesis also play important roles in ferroptosis. The proper functioning of mitochondria directly affects the cell's sensitivity to ferroptosis.

5. Iron in Ferroptosis

Iron plays a central role in the ferroptosis process. Enzymes involved in phospholipid peroxidation, such as LOXs and POR, are iron-dependent. Furthermore, iron in the Fenton reaction can catalyze further reactions of PLOOHs, exacerbating the ferroptosis process. Regulation of intracellular iron homeostasis is achieved through mechanisms involving iron regulatory proteins (IRP1 and IRP2), transferrin, and heme oxygenase 1 (HO-1). Both excess and deficiency of iron can influence the cell's sensitivity to ferroptosis.

Figure 1. Pathways and mechanisms of ferroptosis suppression. (Jiang X, et al., 2021)

6. GPX4-Independent Ferroptosis Regulatory Mechanisms

In addition to the classical GPX4-dependent mechanisms, research has identified GPX4-independent ferroptosis regulatory mechanisms:

• NAD(P)H/FSP1/CoQ10 Axis: Ferroptosis suppressor protein 1 (FSP1) serves as an alternative system to GPX4. It can reduce ubiquinone (CoQ10) to mitigate the effects of lipid free radicals, thereby protecting cells from ferroptosis.
• GCH1/BH4/DHFR Axis: GTP cyclohydrolase 1 (GCH1) protects cells from ferroptosis through its metabolites tetrahydrobiopterin (BH4) and dihydrobiopterin (BH2). BH4 not only acts directly as an antioxidant but also participates in the synthesis of CoQ10, helping to maintain the cellular redox state.

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Nine Important Research Directions

Interpretation of Nine Major Research Directions

To assist medical researchers in comprehensively understanding and quickly grasping the latest developments in ferroptosis research, we have outlined nine key research directions along with corresponding case studies and research strategies:

1. Relationship Between Metabolic Pathways and Ferroptosis

Case Study: Regulation of Ferroptosis Sensitivity by the Distal Cholesterol Synthesis Pathway

7-Dehydrocholesterol (7-DHC), a crucial metabolic intermediate in cholesterol biosynthesis, is synthesized by sterol C5-desaturase (SC5D) and further converted to cholesterol by 7-DHC reductase (DHCR7). While 7-DHC-derived oxysterols have shown neurotoxic effects, the biological function of 7-DHC itself remains unclear. A study using whole-genome CRISPR-Cas9 screening revealed that several enzymes in the distal cholesterol biosynthesis pathway play key and opposing roles in ferroptosis regulation. Specifically, cells manage ferroptosis by modulating 7-DHC levels. MSMO1, CYP51A1, EBP, and SC5D were identified as potential inhibitors of ferroptosis, whereas DHCR7 was found to promote it. This study highlights the mechanism by which key enzymes in distal cholesterol synthesis influence ferroptosis sensitivity through 7-DHC regulation, suggesting that targeting 7-DHC levels could offer new therapeutic strategies for cancer and ischemia-reperfusion injury.

Research Strategy:

• Utilize whole-genome CRISPR-Cas9 screening to identify enzymes involved in distal cholesterol biosynthesis and their role in ferroptosis regulation.
• Investigate the relationship between 7-DHC levels and ferroptosis sensitivity.
• Determine the mechanisms by which MSMO1, CYP51A1, EBP, and SC5D act as ferroptosis inhibitors and DHCR7 as a promoter.

Figure 2. Analysis of the role of 7-DHC in the suppression of ferroptosis across various HEK293T cell models and conditions. (Li Y, et al., 2024)

2. Organelle Interactions and Ferroptosis

Case Study: Mitochondrial-Associated Membranes (MAMs) Regulate Ferroptosis

Mitochondrial-associated membranes (MAMs) are special structures connecting the endoplasmic reticulum and mitochondria, playing central roles in calcium signaling, lipid transfer, and cell death processes. However, their specific role in ferroptosis remains unexplored. Researchers using siRNA to knock out SERCA2 found that this inhibited erastin-induced ferroptosis. Further studies indicated that MAMs regulate ferroptosis through calcium ion transport and lipid remodeling, with Ca2+ (especially ER Ca2+ storage) being crucial in ferroptosis execution. Additionally, CGI1746 was found to inhibit ferroptosis via the sigma-1 receptor (σ1R). This research provides new insights into MAMs' functions and suggests that σ1R within MAMs may be a potential therapeutic target for excessive ferroptosis.

Research Strategy:

• Study the role of MAMs in calcium ion transport and lipid remodeling.
• Investigate the impact of ER calcium storage on ferroptosis execution.
• Identify compounds targeting MAMs (e.g., CGI1746) and their mechanisms.

Figure 3. CGI1746 blocks calcium transfer from the endoplasmic reticulum to mitochondria during ferroptosis. (Zhang Z, et al., 2024)

3. Targeted Inhibition of Key Ferroptosis Enzymes

Case Study: ACSL4 as a Therapeutic Target for Ferroptosis

Ferroptosis plays a crucial role in tumor suppression, with tumor suppressors like p53 and BAP1 promoting ferroptosis by downregulating SLC7A11. AS-252424 (AS), a selective PI3Kγ inhibitor, has been shown to effectively inhibit lipid peroxidation and ferroptosis in human and mouse cells, despite its known activity against reactive oxygen species (ROS). ACSL4, a lipid metabolism enzyme that promotes ferroptosis, lacks specific inhibitors to date. This study highlights ACSL4 as a key pharmacological target for treating ferroptosis-related diseases and suggests potential therapeutic strategies.

Research Strategy:

• Screen kinase inhibitor libraries to find specific ACSL4 inhibitors (e.g., AS-252424).
• Investigate the effects of ACSL4 inhibitors on lipid peroxidation and ferroptosis.
• Explore the application of ACSL4 inhibition in treating ferroptosis-related diseases.

Figure 4. As inhibits ferroptosis across various cancer and neuronal cell types. (Huang Q, et al., 2024)

4. Tumor Microenvironment and Ferroptosis

Case Study: Neutrophil Ferroptosis Impact on Gastric Cancer Immunotherapy

The tumor microenvironment (TME) refers to the local biological environment of solid tumors and has emerged as an attractive target for cancer therapy. In gastric cancer (GC), neutrophils within the TME undergo ferroptosis, releasing oxidized lipids that inhibit T-cell activity. Enhanced photodynamic therapy (PDT) using di-iodinated IR780 (Icy7) significantly increased ROS production, which triggered neutrophil ferroptosis in the TME. This finding provides a theoretical basis for understanding GC's TME and improving immunotherapy effectiveness.

Research Strategy:

• Analyze neutrophil ferroptosis status in the gastric cancer TME.
• Study the impact of neutrophil ferroptosis on the immune microenvironment.
• Explore the synergistic effects of enhanced PDT and inhibition of neutrophil ferroptosis.

Figure 5. Inhibition of neutrophil ferroptosis enhances cd8+ t cell function. (Zhu X, et al., 2024)

5. Application of Nanotechnology in Ferroptosis Therapy

Case Study: Mitochondria-Targeted Prodrug Nanocapsules Enhancing Ferroptosis Therapy

Ferroptosis in tumor cells is limited by cellular defense systems, such as GPX4 and CoQH2 systems. Researchers designed a defined single-molecule nanocapsule (QSSP) combining DHODH inhibitors (QA) and a triphenylphosphine moiety linked by disulfide bonds. Upon entering cancer cells, the acidic environment triggered QSSP disassembly, releasing free prodrug molecules. Lipophilic cationic prodrugs accumulated in mitochondria, where glutathione levels were reduced by thiol-disulfide exchange, leading to GPX4 inactivation and ferroptosis promotion. QA released from ester hydrolysis further disrupted mitochondrial defenses, inducing strong ferroptosis. This subcellular-targeted nanocapsule offers important references and methods for ferroptosis-based cancer therapies.

Research Strategy:

• Design mitochondria-targeted prodrug nanocapsules (e.g., QSSP).
• Study the mechanism by which nanocapsules disrupt ferroptosis defense systems (GPX4, CoQH2).
• Evaluate the effectiveness of nanocapsules in cancer therapy.

Figure 6. Molecular mechanism of QSSP-induced ferroptosis. (Liu N, et al., 2024)

6. Role of Polyunsaturated Fatty Acids in Ferroptosis

Case Study: Phospholipids with Two Polyunsaturated Fatty Acid Tails (PL-PUFA2s) Promote Ferroptosis

Phospholipids with a single polyunsaturated fatty acid tail (PL-PUFA1s) are known to drive ferroptosis, while phospholipids with two polyunsaturated fatty acid tails (PL-PUFA2s) have been less studied. Researchers evaluated 20 human cancer cell lines' sensitivity to RSL3-induced ferroptosis, identifying a rare lipid PC-PUFA2 that plays a critical role in ferroptosis. Further analysis showed PC-PUFA2's crucial role in regulating mitochondrial homeostasis and ferroptosis, highlighting the mechanism of free fatty acids in ferroptosis regulation. This study suggests that PC-PUFA2 could serve as a diagnostic and therapeutic target for ferroptosis, opening new avenues for treating neurodegenerative diseases and cancer.

Research Strategy:

• Analyze the impact of different phospholipids on ferroptosis.
• Investigate the molecular mechanisms by which PL-PUFA2s promote ferroptosis.
• Explore the potential of regulating PL-PUFA2 levels to influence ferroptosis.
• Assess the potential of PL-PUFA2s as diagnostic and therapeutic targets.

Figure 7. PC-PUFA2s induce ferroptosis. (Qiu B, et al., 2024)

7. Role of DNA Repair Enzymes in Ferroptosis

Case Study: Inhibition of APE1 Promotes Ferroptosis in Hepatocellular Carcinoma Cells

Ongoing oxidative stress during ferroptosis upregulates APE1 expression, enhancing tumor cell resistance to ferroptosis. The specific role of APE1 in hepatocellular carcinoma (HCC) ferroptosis remains unclear. Researchers used H2DCFDA and C11-BODIPY 581/591 dyes to evaluate ROS and lipid peroxidation levels in control and APE1-KD cells, finding that APE1 deficiency significantly increased HCC cells' sensitivity to ferroptosis inducers erastin and RSL3. Mechanistic studies showed that APE1 regulates HCC ferroptosis through its redox function rather than DNA repair. This research reveals APE1's critical role in regulating ferroptosis in HCC cells, providing new targets and strategies for HCC treatment.

Research Strategy:

• Screen DNA repair enzymes involved in ferroptosis regulation.
• Investigate the specific mechanisms of these enzymes in ferroptosis.
• Develop inhibitors targeting these enzymes.
• Evaluate the potential of these inhibitors in tumor therapy.

Figure 8. Inhibition of APE1 promotes ferroptosis in liver cancer cells. (Du Y, et al., 2024)

8. Role of Receptor Tyrosine Kinases in Ferroptosis and Immunotherapy

Case Study: Targeting MerTK Enhances Ferroptosis and Immune Response in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) incidence is rising globally, and while immune checkpoint inhibitors (ICIs) have shown some success, resistance to PD-1/PD-L1 inhibitors remains a significant issue. Researchers found that MerTK, a proto-oncogene tyrosine kinase, is highly expressed in HCC and contributes to resistance by limiting tumor cell ferroptosis and increasing myeloid-derived suppressor cell infiltration. Studies using gene-modified mouse models and Hepa1-6 subcutaneous tumor models showed that anti-PD-L1 antibodies were ineffective in controlling resistant tumors. MerTK could serve as a biomarker for HCC and provide new strategies for overcoming immune therapy resistance.

Research Strategy:

• Identify receptor tyrosine kinases involved in ferroptosis regulation.
• Study the impact of these kinases on ferroptosis and immune responses.
• Develop inhibitors targeting these kinases.
• Evaluate the combined effect of these inhibitors with immune checkpoint inhibitors.

Figure 9. Suppressing SLC7A11 Enhances Sensitivity to Anti-PD-L1 Therapy in Liver Cancer. (Wang S, et al., 2024)

9. Epigenetic Regulation in Ferroptosis

Case Study: GAS41 Regulates Ferroptosis by Modulating NRF2 Transcription

Hepatocellular carcinoma (HCC) is a major global health issue with rising incidence and mortality. Although ICIs such as anti-CTLA4 and anti-PD-1/PD-L1 have shown effectiveness, resistance is common and response rates are suboptimal. Identifying effective resistance targets is crucial. Researchers found that MerTK, a proto-oncogene tyrosine kinase, is highly expressed in HCC and limits tumor cell ferroptosis. Using mouse tumor models, the study demonstrated that anti-PD-L1 antibody treatment failed to produce antitumor effects in resistant cases. This suggests MerTK as a major mechanism of PD-1/PD-L1 blockade resistance and its role in creating an immune-suppressive TME. MerTK could thus serve as a biomarker and provide new strategies for HCC immunotherapy.

Research Strategy:

• Identify epigenetic factors involved in ferroptosis regulation.
• Study the impact of these factors on ferroptosis-related gene expression.
• Explore targeting these epigenetic factors to regulate ferroptosis.
• Assess the potential of this regulatory strategy in cancer therapy.

Figure 10. GAS41's Interaction with NRF2 is Essential for its Transcriptional Function. (Wang Z, et al., 2024)

List of Related Genes

Related Target or Gene List