Disulfidptosis is a form of regulated cell death driven by abnormal intracellular disulfide accumulation and collapse of the actin cytoskeleton. It typically occurs in cells with high SLC7A11 expression under conditions of restricted glucose utilization and insufficient NADPH supply, reflecting the metabolic connection among cystine uptake, cellular reducing capacity, and actin-cytoskeleton homeostasis.

Keywords: disulfidptosis; SLC7A11; cystine; NADPH; disulfide stress; actin cytoskeleton; F-actin; glucose restriction; tumor metabolism

1 Basic Characteristics of Disulfidptosis

1.1 Definition of Disulfidptosis

Disulfidptosis refers to a process in which cells with insufficient reducing capacity cannot efficiently process imported cystine, resulting in abnormal accumulation of cystine and other disulfides and inducing the formation of aberrant disulfide bonds in actin-cytoskeleton-associated proteins. This ultimately causes contraction of the F-actin network, cytoskeletal collapse, and cell death. Its core execution event is not a Caspase cascade, lipid peroxidation, or Gasdermin pore formation, but structural damage to the cytoskeleton caused by disruption of disulfide-bond homeostasis.

1.2 Conditions Required for Disulfidptosis

(1) High SLC7A11 Expression

SLC7A11 and SLC3A2 together form the system Xc⁻ cystine/glutamate antiporter. High SLC7A11 expression enhances extracellular cystine uptake and supplies substrates for cysteine and glutathione synthesis. However, it also increases the demand for NADPH during cystine reduction, making cells highly dependent on glucose metabolism and the supply of reducing equivalents.

(2) Insufficient NADPH Supply

Glucose deprivation, inhibition of glucose transport, or restriction of the pentose phosphate pathway can all reduce cytosolic NADPH levels and impair cystine reduction, glutathione regeneration, and the thioredoxin system, thereby promoting intracellular disulfide accumulation.

(3) Persistent Presence of Extracellular Cystine

Glucose deprivation alone is not equivalent to disulfidptosis. A substantial disulfide burden is more likely to develop only when cells with high SLC7A11 expression continuously import cystine while lacking sufficient reducing capacity. Removal of extracellular cystine, reduction of SLC7A11 expression, or inhibition of cystine uptake can alleviate this process.

Figure 1. SLC7A11-mediated cystine metabolic imbalance and the mechanism of disulfidptosis induction

1.3 Disulfidptosis and Oxidative Stress

Disulfidptosis is a form of cell death associated with disruption of redox homeostasis, but it cannot be simply attributed to an overall increase in reactive oxygen species. Its key alterations are NADPH insufficiency, disulfide accumulation, and abnormal remodeling of protein disulfide bonds. Therefore, changes in ROS, GSH, or GSSG can only serve as supplementary indicators and cannot independently confirm disulfidptosis.

2 Core Molecular Mechanisms of Disulfidptosis

2.1 SLC7A11-Mediated Cystine Uptake

SLC7A11 transports extracellular cystine into cells while exporting intracellular glutamate. Under normal conditions, imported cystine is reduced to cysteine and used for the synthesis of glutathione, proteins, and other sulfur-containing metabolites. When SLC7A11 is persistently overexpressed, cystine influx increases, accompanied by an increased cellular requirement for NADPH and other reducing systems.

2.2 NADPH Depletion and Disulfide Accumulation

When glucose is sufficient, NADPH generated through the pentose phosphate pathway supports cystine reduction and maintains the reduced state of the glutathione and thioredoxin systems. When glucose utilization is restricted, cystine continuously entering the cell cannot be fully converted into cysteine, resulting in the accumulation of cystine, γ-glutamylcystine, and other disulfides and thereby causing disulfide stress.

2.3 Abnormal Disulfide-Bond Formation in the Actin Cytoskeleton

Disulfide stress can promote the formation of abnormal intramolecular or intermolecular disulfide bonds between cysteine residues in actin and actin-regulatory proteins. These changes disrupt actin-filament polymerization, branching, bundling, and tension distribution, ultimately producing abnormal stress-fiber contraction, F-actin aggregation, and collapse of the overall cytoskeletal network.

2.4 The Rac-WRC-Arp2/3 Regulatory Axis

Rac small GTPases can activate the WAVE regulatory complex (WRC), which subsequently promotes Arp2/3-dependent branched actin polymerization. An active Rac-WRC-Arp2/3 network provides an abundant cytoskeletal structural basis for abnormal disulfide crosslinking. Impaired WRC function can reduce the sensitivity of some cells to disulfidptosis, whereas enhanced Rac signaling may aggravate cytoskeletal contraction and cell death.

2.5 Regulation by Cellular Reducing Systems

The glutathione and thioredoxin systems jointly maintain the balance between protein thiols and disulfide bonds. When glutathione-regeneration capacity declines or thioredoxin reductase is inhibited, the ability to remove abnormal disulfide bonds is further reduced, which can intensify disulfide stress and cytoskeletal damage under glucose-restricted conditions.

3 Cellular and Biochemical Features of Disulfidptosis

3.1 Cellular Morphological Changes

Cells undergoing disulfidptosis commonly exhibit rapid contraction, reduced spreading area, retraction of cell edges, loss of matrix adhesion, and cell detachment. Abnormal F-actin contraction and network collapse are representative structural features. However, cell rounding and contraction can also occur after apoptosis, adhesion injury, or treatment with cytoskeletal drugs and therefore cannot serve as independent diagnostic evidence.

3.2 Metabolic Changes

Disulfidptosis may involve decreased NADPH, an increased NADP⁺/NADPH ratio, increased intracellular cystine and other disulfides, and altered GSH/GSSG balance. These metabolic indicators should be analyzed together with SLC7A11 dependence, rescue by reducing agents, and changes in the actin cytoskeleton.

3.3 Changes in Protein Disulfide Bonds

SDS-PAGE or Western blotting under nonreducing conditions may reveal altered migration, high-molecular-weight aggregates, or smeared signals in certain actin-cytoskeleton-associated proteins. Attenuation of these signals after the addition of DTT, β-Mercaptoethanol, or other reducing agents supports the formation of abnormal disulfide bonds, but interpretation still requires analysis of specific proteins and functional results.

3.4 Changes in F-Actin Structure

Fluorescent phalloidin or Actin-Tracker probes can be used to observe F-actin structures. Disulfidptosis commonly manifests as reduced lamellipodia, abnormal aggregation of stress fibers, contraction of actin bundles, and reduced cytoskeletal continuity. Cell area, circularity, total F-actin intensity, fiber orientation, and signal distribution at the cell edge can be further quantified.

4 Differences Between Disulfidptosis and Other Forms of Cell Death

5 Experimental Induction of Disulfidptosis

5.1 Selection of Cell Models

Cells with high endogenous SLC7A11 expression and sensitivity to glucose restriction should be prioritized, with SLC7A11-low-expression, overexpression, knockdown, or knockout controls included. In addition to transcript levels, SLC7A11 protein expression, plasma-membrane localization, cystine-uptake capacity, and the cellular ability to maintain NADPH should be examined.

5.2 Glucose-Starvation Models

Cells can be transferred into glucose-free or low-glucose medium while maintaining generally consistent levels of cystine and other major nutrients. Glucose-concentration and treatment-time gradients should be included to avoid generalized ATP depletion, endoplasmic reticulum stress, or other secondary forms of cell death caused by prolonged nutrient deprivation.

5.3 Glucose-Utilization Inhibition Models

Glucose-transporter inhibitors can reduce cellular glucose uptake and pentose-phosphate-pathway-derived NADPH production and can induce disulfidptosis in SLC7A11-high cells. Although 2-Deoxy-D-Glucose inhibits glycolysis, its phosphorylated metabolite, 2-DG-6-P, can enter the pentose phosphate pathway and support NADPH production; in some models, it alleviates glucose-starvation-induced disulfidptosis. Therefore, 2-Deoxy-D-Glucose should not be used as a direct tool for restricting NADPH supply or inducing disulfidptosis.

5.4 Reducing-System Intervention Models

Inhibition of glutathione regeneration or the thioredoxin-reduction system under glucose-restricted conditions can enhance disulfide accumulation. Such interventions may also cause extensive oxidative damage, so it is necessary to verify whether cell death remains dependent on SLC7A11, extracellular cystine, and abnormal disulfide bonds in actin.

6 Experimental Identification of Disulfidptosis

6.1 Cell-Death Detection

Cellular metabolic activity, ATP levels, LDH release, and PI or 7-AAD staining can be used to assess cellular injury and death. A single viability indicator only demonstrates cellular damage and cannot determine the form of cell death. Metabolic changes, protein disulfide bonds, and F-actin structures should be analyzed simultaneously.

6.2 Validation of SLC7A11 and Cystine Dependence

SLC7A11 knockdown, knockout, or removal of extracellular cystine should alleviate glucose-restriction-induced cell death. It should be noted that under glucose-sufficient conditions, inhibition of SLC7A11 may promote ferroptosis by reducing GSH synthesis. Therefore, the results of SLC7A11 intervention must be interpreted together with the nutrient environment and lipid-peroxidation status.

6.3 Detection of NADPH and Sulfur-Containing Metabolites

NADPH, the NADP⁺/NADPH ratio, cystine, cysteine, GSH, and GSSG should be measured. Decreased NADPH and cystine accumulation support the presence of disulfide stress, but these findings must still be combined with F-actin collapse, increased protein disulfide bonds, and causal rescue experiments.

6.4 Rescue With Reducing Agents

Reducing agents such as DTT or β-Mercaptoethanol can decrease the disulfide burden and alleviate cell death and are important tools for validating disulfide dependence. Reducing agents broadly affect cystine and protein disulfide bonds in the culture system, so reducing-agent-only treatment groups and concentration gradients should be included. TCEP is more suitable for lysates, protein samples, and comparison between reducing and nonreducing electrophoresis conditions.

6.5 Exclusion of Other Forms of Cell Death

Caspase inhibitors, ferroptosis inhibitors, iron chelators, and necroptosis inhibitors can be used separately, together with detection of Cleaved Caspase-3, lipid peroxidation, p-MLKL, and related indicators. Failure of these inhibitors to provide effective rescue can only help exclude major execution pathways and cannot replace the positive mechanistic evidence required to identify disulfidptosis.

7 Regulatory Factors of Disulfidptosis

7.1 BAP1-SLC7A11 Regulation

BAP1 can reduce cystine uptake by lowering SLC7A11 expression, thereby decreasing the disulfide burden under glucose-deficient conditions. This effect may be opposite to its outcome in ferroptosis, demonstrating that the influence of SLC7A11 on different forms of cell death depends on glucose availability, NADPH status, and the lipid-antioxidant environment.

7.2 Endoplasmic Reticulum Stress Responses

Glucose deficiency and disulfide accumulation can simultaneously induce endoplasmic reticulum stress. A moderate unfolded protein response may help cells restore protein homeostasis, whereas persistent stress or inhibition of adaptive responses may enhance cell death. The specific effects are strongly dependent on cell type and treatment duration.

7.3 Cell Density and Metabolic State

Cell density can affect glucose consumption, nutrient competition, cell adhesion, actin tension, and SLC7A11 stability and thereby alter sensitivity to disulfidptosis. Seeding density, culture duration, serum conditions, and medium volume should be standardized to avoid misinterpreting differences in culture conditions as effects of a specific pathway.

8 Disease Research and Targeting Potential

8.1 Tumors With High SLC7A11 Expression

Some tumors increase SLC7A11 expression to enhance cysteine and GSH supply and resist ferroptosis and oxidative damage. However, this also creates strong dependence on glucose and NADPH supply. Restricting glucose utilization may therefore selectively increase disulfide stress and cytoskeletal damage in these tumor cells.

8.2 Combination Intervention Strategies

Inhibition of glucose transport or utilization can be combined with intervention in the thioredoxin-reduction system, regulation of the Rac-WRC-Arp2/3 axis, or other metabolic inhibition strategies. Combination regimens need to exclude generalized energy depletion, extensive oxidative damage, and nonspecific actin toxicity and should evaluate the tolerance of normal tissues to glucose restriction.

8.3 Research on Non-Tumor Diseases

The relationships between disulfidptosis and ischemia, neurological injury, and other metabolism-related stress disorders require further mechanistic validation. Observation of increased SLC7A11 or changes in related gene expression alone cannot demonstrate disulfidptosis in tissues. Direct evidence of disulfide accumulation, abnormal protein disulfide bonds, and F-actin collapse is also required.

9 Products

9.1 Products for Disulfidptosis Model Establishment and Disulfide-Bond Rescue

9.2 Products for SLC7A11 and Redox-State Detection

9.3 Products for F-Actin and Actin-Cytoskeleton Research

9.4 Control Products for Inducing and Excluding Other Forms of Cell Death

Reliable disulfidptosis research should simultaneously demonstrate glucose or NADPH insufficiency, cystine-dependent disulfide accumulation, abnormal protein disulfide-bond formation, and F-actin collapse. A complete evidence chain should be established through rescue with reducing agents and experiments excluding other forms of cell death.

For more related articles, please see below:

[1] Experiments for the morphological evaluation of cell death

[2] Experiments on biochemical analysis of cell death

[3] Staining Principles and Methods for Cell Death, Proliferation, and Metabolic Viability

[4] Cytokines and Inflammation

[5] Microscopic morphologic observation of apoptosis

[6] Cellular Copper Death Assay

[7] Apoptosis Signaling Pathways and Regulated Cell Death Research

[8] Molecular Basis of Ferroptosis-Immune Metabolism Crosstalk