Copper is an essential trace element in animals and is widely distributed in tissues including muscle, bone, and liver (Festa and Thiele, 2011). Dietary copper is primarily absorbed through the gastrointestinal tract (Xiong et al., 2023), mainly in the small intestine. Copper uptake across the enterocyte apical membrane relies on copper transporter 1 (CTR1), which facilitates the entry of Cu⁺ (Nose et al., 2006). Alternatively, copper can be absorbed as Cu²⁺ via divalent metal transporter 1 (DMT1) and is subsequently reduced to Cu⁺ by cellular sulfhydryl groups (Sa et al., 2018; Gunshin et al., 1997). Copper is then exported across the basolateral membrane via the ATPase copper transporting alpha (ATP7A) protein (Wang et al., 2012). The targeted delivery of intracellular copper relies on multiple metallochaperone systems:(a) Cytochrome c oxidase copper chaperone COX17 (Cox17) transports copper to mitochondria and participates in cytochrome c oxidase assembly (Amaravadi et al., 1997); (b)The copper chaperone for superoxide dismutase (CCS) delivers copper to superoxide dismutase 1 (SOD1) in the cytoplasm and activates its antioxidant function (Culotta et al., 1997); (c) Antioxidant protein 1 (ATOX1) cooperates with P-type ATPases to deliver copper to the Golgi network for subsequent excretion or transmembrane transport (Lutsenko et al., 2007). Besides, other companion proteins send copper to specific places inside the cell (Banci et al., 2010). In the portal circulation, copper is transported to the liver primarily bound to serum proteins like albumin. Hepatocytes store excess copper using metallothioneins (MT1 and MT2) (Ge et al., 2022). Biliary copper excretion is mediated by ATPase copper transporting beta (ATP7B), a protein located on the bile duct membrane, and serves as a key pathway for maintaining systemic copper homeostasis (Roelofsen et al., 2000; Polishchuk et al., 2014). Intracellular free copper levels are tightly controlled and kept extremely low by the homeostatic system to prevent toxic reactions caused by copper accumulation (Ge et al., 2022; Lutsenko, 2010; Rae et al., 1999; Kim et al., 2008) (Figure 1).

Intracellular copper ions primarily bind to both copper-specific and non-copper-specific proteins for functional regulation and homeostasis maintenance. The binding mechanisms between these two types of proteins differ significantly. Copper proteins incorporate copper ions through highly specific and stable coordination—such as binding to histidine or cysteine residues—to enable enzymatic activity or support structural functions (Adman et al., 1991). In contrast, non-copper proteins (such as metallothionein) participate in copper metabolism through non-specific binding, buffering storage, and transport regulation (Kodama et al., 2012). These proteins play an auxiliary role in maintaining dynamic copper homeostasis within the cell. Additionally, copper chaperone proteins (e.g., ATOX1, CCS) are responsible for the targeted and precise delivery of copper ions (Lippard and Berg, 1994). Meanwhile, small molecules like glutathione help buffer intracellular copper levels and mitigate oxidative toxicity through reversible binding (Que et al., 2008; Solomon et al., 1996). Together, these mechanisms form a multi-layered and dynamically balanced copper homeostatic network, which protects cells from copper-induced toxicity while supporting essential biological processes that depend on copper.

Copper is an essential trace element in living organisms and is widely present in diverse cells—from bacteria to humans (Kim et al., 2008). It serves as a cofactor for many key enzymes and plays a vital role in maintaining cellular homeostasis, a function well supported by scientific studies (Ge et al., 2022). Specifically, copper participates in regulating several fundamental cellular processes, such as mitochondrial respiration, antioxidant defense, and the biosynthesis of hormones, neurotransmitters, and pigments (Lippard and Berg, 1994; Que et al., 2008; Solomon et al., 1996). For example, copper acts as a key cofactor for mitochondrial cytochrome c oxidase and is essential for meeting the energy demands of rapidly proliferating cells (Lopez et al., 2019); It also serves as an essential cofactor for Cu/Zn SOD1, supporting cellular antioxidant defense mechanisms (Rae et al., 1999). It is noteworthy that despite its strong redox activity, copper rarely exists in a free form inside cells. Studies indicate that the intracellular concentration of free Cu²⁺ is extremely low under normal conditions, with an estimated upper limit of only 10^–18 M (Lippard, 1999). This strict homeostatic regulation is essential for understanding copper’s role in pathological conditions.

Notably, dysregulation of copper metabolism can significantly impact physiological functions. Copper metabolism disorder, which involves either excessive copper accumulation or improper transport, is detrimental to humans (Scheiber et al., 2013). This phenomenon is particularly evident in tumor cells, which exhibit a higher demand for copper than normal cells (Denoyer et al., 2015). To support the energy and biosynthetic demands of rapid proliferation, copper levels are significantly elevated in tumor cells (Chen and Pervaiz, 2010). Clinical studies have revealed abnormally elevated copper levels in both tumor tissues and serum across multiple cancer types, such as thyroid (Baltaci et al., 2017), breast (Kuo et al., 2002; Feng et al., 2012), lung (Lossow et al., 2021; Jin et al., 2011), pancreatic (Lener et al., 2016), gallbladder (Basu et al., 2013), and colorectal cancers (Gupta et al., 1993). This elevation is strongly associated with poor patient prognosis.

Tumor cells adapt their copper metabolism in multiple ways to support malignant proliferation. First, many cancer cells exhibit high expression of CTR1, leading to significantly enhanced copper uptake (Zimnicka et al., 2014). Second, cancer cells modify the expression and localization of copper chaperone proteins to preferentially deliver copper ions to enzymes involved in proliferation and angiogenesis, thereby promotes these key processes (Suwara and Hartman, 2025). Moreover, increased intracellular copper levels activate multiple signaling pathways, such as mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) and B-rapidly accelerated fibrosarcoma (BRAF),which stimulate cell proliferation, promote angiogenesis via vascular endothelial growth factor (VEGF) activation (Pan et al., 2002), and enhance tumor invasion and metastasis by inducing epithelial-mesenchymal transition (EMT) (Zhang et al., 2025). However, excessive copper accumulation acts as a “double-edged sword” in tumors. On one hand, copper promotes tumor growth; on the other hand, excess copper induces high levels of ROS through the Fenton reaction, leading to severe oxidative damage in proteins, lipids, and DNA (Banci et al., 2010; Basu et al., 2013). Furthermore, high copper levels can directly or indirectly modulate key apoptotic pathway proteins, leading to programmed cell death (Guo et al., 2025).

Disrupted copper metabolism also significantly influences immune regulation within the tumor microenvironment (TME). TME is a complex ecosystem composed of tumor cells, extracellular matrix (ECM), innate and adaptive immune cells, inflammatory cells, tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), tumor-associated neutrophils, myeloid-derived suppressor cells (MDSCs), endothelial cells, microvascular networks, as well as various cytokines and chemokines (Wu et al., 2022). These components interact dynamically to form an immunosuppressive microenvironment (Yang et al., 2021; Mo et al., 2021). Interestingly, studies indicate that elevated copper levels in the TME are positively correlated with increased expression of the programmed death-ligand 1(PD-L1) (Cobine and Brady, 2022). PD-L1 binds to programmed cell death protein 1(PD-1) on T cells and suppresses downstream signaling pathways such as rat sarcoma/mitogen-activated protein kinase (Ras/MAPK) and phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT), leading to T cell apoptosis and exhaustion, thereby facilitating tumor immune escape (Kciuk et al., 2023).

In this complex microenvironment, the functional state of macrophages plays a particularly critical role. Macrophages can be categorized into three main types: the unactivated M0 type, the classically activated pro-inflammatory M1 type, and the alternatively activated anti-inflammatory M2 type (Chaintreuil et al., 2023). M1 macrophages inhibit tumor growth by secreting cytokines like tumor necrosis factor-alpha (TNF-α), while M2 macrophages promote tumor progression and angiogenesis through factors such as interleukin-6(IL-6), interleukin-10(IL-10), and VEGF (54). Studies indicate that excess copper markedly suppresses macrophage function and promotes polarization toward the M2 phenotype, thereby enhancing immunosuppression (Zhao and Zhao, 2019; Tang et al., 2023). Moreover, an effective anti-tumor response by immune cells relies on normal mitochondrial respiratory function (Chang et al., 2022). However, copper overload disrupts mitochondrial function, leading to impaired energy metabolism and increased oxidative stress (Roberts et al., 2008; Sokol et al., 1994; Gu et al., 2000). Consequently, immune cells lose their capacity to effectively recognize and attack tumor cells, resulting in a weakened anti-tumor immune response (Chang et al., 2022).

In summary, cancer cells display distinct copper metabolic characteristics, primarily marked by increased intracellular copper levels and a strong reliance on copper-dependent oncogenic mechanisms (Yang et al., 2023). The link between copper metabolism and tumor development has been widely investigated, with substantial evidence supporting copper’s key role in tumor initiation and progression (Ishida et al., 2013). At the metabolic level, copper promotes metabolic reprogramming in tumor cells by regulating oxidative phosphorylation and energy metabolism, thereby supporting their rapid proliferation. At the molecular level, it elevates cellular oxidative stress and stimulates angiogenesis, thereby accelerating tumor progression. Concurrently, dysregulated copper metabolism is also a consequence of the malignant phenotype in cancer cells. Tumor cells often upregulate the CTR1 to meet their elevated copper demand (Zimnicka et al., 2014). Additionally, hypoxia-inducible factor-1α(HIF-1α) indirectly stabilizes and activates the transcription of multiple copper metabolism-related genes, such as those regulating CTR1 (Su et al., 2022). This mechanism further enhances copper accumulation in tumor cells and establishes a positive feedback loop that continuously drives malignant progression.

Recently, a novel form of copper-dependent cell death, reliant on mitochondrial respiration, has been identified and termed “cuproptosis” (Tsvetkov et al., 2022). It describes how copper affects cells through signaling pathways and both enzymatic and non-enzymatic activities (Ge et al., 2022). Cuproptosis can be controlled by using genes or drugs to target proteins related to copper homeostasis. During this process, copper enhances mitochondria-dependent energy metabolism and induces ROS accumulation, resulting in cytotoxicity—a mechanism now referred to as “cuproptosis” (Ge et al., 2022). Cell death involves complex signaling pathways and molecular mechanisms (Tang et al., 2019), which lead to changes in proteins and lipids (Carneiro and El-Deiry, 2020). The main types of cell death include necrosis (Weinlich et al., 2017), pyroptosis (Bergsbaken et al., 2009), ferroptosis (Dixon et al., 2012) and other forms. Previous studies have shown that elesclomol (ES) induces apoptosis through the ROS-dependent mechanism (Hasinoff et al., 2014; Kirshner et al., 2008). However, the cell death caused by ES does not involve the breakdown of the caspase-3, a marker of apoptosis,and not activate caspase-3 (Elmore, 2007). The killing potential of ES was not affected when cells were knocked out of apoptosis effector or treated with other inhibitors of known cell death mechanisms, suggesting that the cell death pathway induced by copper ionophores is different from known cell death pathways (Tsvetkov et al., 2022).

Studies have demonstrated that cuproptosis is closely linked to mitochondrial protein lipoylation. Protein lipoylation is a key post-translational modification involving the covalent attachment of an acyl group (e.g., acetyl, propionyl, succinyl, etc.) to specific amino acid residues, most commonly the ε-amino group of lysine (Resh, 2016). The modification is known to occur only in four key metabolic enzymes: dihydrolipoamide branched-chain transacylase E2 (DBT), dihydrolipoamide-S-succinyltransferase (DLST),dihydrolipoamide-S-acetyltransferase (DLAT) and glycine cleavage system H protein (GCSH) (Solmonson and DeBerardinis, 2018; Rowland et al., 2018). All these enzymes play essential roles in directing carbon sources into the tricarboxylic acid cycle (TCA cycle): DBT, as part of the branched-chain ketoacid dehydrogenase complex, catalyzes the conversion of branched-chain α-ketoacids to acyl-CoA through lipoylation (Lin et al., 2024); DLST promotes the transformation of α-ketoglutarate to succinyl-CoA within the α-ketoglutarate dehydrogenase complex (Lin et al., 2024); DLAT, a core component of the pyruvate dehydrogenase complex, converts pyruvate to acetyl-CoA (La et al., 2022); GCSH serves as a lipoyl carrier protein and enhances enzymatic activity by modifying DLST and DLAT. Together, they regulate the generation of TCA cycle intermediate metabolites (Mukha et al., 2022). These lipoylation modifications are crucial for sustaining normal enzyme function (Rowland et al., 2018), and ferredoxin 1(FDX1) is considered a key upstream regulator of this process.

FDX1 is a small iron-sulfur cluster protein that transfers electrons from Nicotinamide Adenine Dinucleotide Phosphate (NADPH) to the mitochondrial cytochrome P450 system via ferredoxin reductase (FDXR). It participates in key biological processes including steroid synthesis, cholesterol metabolism, and bile acid production (Sheftel et al., 2010). In lung adenocarcinoma, downregulation of FDX1 leads to significant alterations in metabolites associated with glucose metabolism, fatty acid oxidation, and amino acid metabolism (Zhang et al., 2021); In gliomas, cellular myelocytomatosis oncogene (c-MYC) upregulates FDX1, thereby inhibiting Microtubule-associated protein 1A/1B-light chain 3 (LC3) mediated mitochondrial autophagy (Guowei et al., 2023). Additionally, FDX1 contributes to the regulation of apoptosis and autophagy in polycystic ovary syndrome (Xing et al., 2023). Notably, FDX1 plays a critical role in cuproptosis: when excess copper ions (particularly in the presence of copper ionophores) enter mitochondria, FDX1 reduces Cu²⁺ to the more reactive Cu⁺. The reduced Cu⁺ directly binds to lipoylated proteins (e.g., DLAT) in the TCA cycle, causing abnormal oligomerization and aggregation, which ultimately leads to serious proteotoxic stress. Simultaneously, this process causes destabilization and functional impairment of iron-sulfur (Fe-S) cluster proteins, ultimately resulting in irreversible cell death.

Cuproptosis strongly depends on copper ionophores—small molecules that bind extracellular copper ions, transport them into cells (especially mitochondria), and thereby induce severe cell death (Hunsaker and Franz, 2019; Oliveri, 2020; Guo et al., 2023). Copper ionophores do not depend on intracellular small molecule chaperones; instead, they directly promote toxic effects by inducing abnormal copper accumulation (Tsvetkov et al., 2022). Additionally, naturally produced copper ionophores have shown strong antimicrobial effects (Raffa et al., 2021; Patteson et al., 2021). So far, several types of copper ionophores have been created, such as disulfide (DSF), 8-hydroxyquinoline (8-HQ), pyrithione, and ES (Kahlson and Dixon, 2022). Among these ionophores, ES is special. It can direct the transport of copper from outside the cell into the mitochondria, which causes copper to build up in the cell. Within the mitochondria, Cu²⁺ is changed to Cu⁺, leading to the production of ROS (Nagai et al., 2012).

Studies on ES further revealed that cell death induced by copper ionophores primarily relies on the continuous accumulation of intracellular copper (Tsvetkov et al., 2022). ES binds to extracellular Cu²⁺ through its nitrogen and sulfur atoms, which act as the primary binding sites (Helsel and Franz, 2015). Specifically, upon Cu²⁺ binding, ES undergoes deprotonation at its N1 and N2 atoms, resulting in the formation of a neutral ES-Cu²⁺ complex (Yadav et al., 2013). After entering the cell, the ES-Cu²⁺ complex is transported to the mitochondria. Within the mitochondria, Cu²⁺ is reduced to Cu⁺ by the action of FDX1. Subsequently, Cu⁺ is released from the complex, while ES continues to be exported, thereby facilitating the repeated shuttling of the ES-Cu²⁺ complex. This cycle causes copper to build up continuously inside the cell. At the same time, ROS levels keep rising steadily. Eventually, the cell’s antioxidant defenses become overwhelmed, triggering oxidative stress (Nagai et al., 2012). Compared with normal cells, cancer cells not only have higher basic levels of ROS and lower antioxidant abilities, but they also show higher oxidative stress levels and rely more on mitochondrial metabolism (Gupte and Mumper, 2009; Trachootham et al., 2009). These characteristics render cancer cells highly sensitive to ES-induced copper toxicity, with their sensitivity being significantly higher than that of healthy cells (Zheng et al., 2022).

However, ES is quickly removed and broken down in the body. This makes it harder for copper ions to enter cancer cells. Despite this, ES has been found to be effective at transporting copper (Nagai et al., 2012), And this high effectiveness might be the reason why we see strong anti-cancer activity in many different types of tumors, such as lung cancer, melanoma, and sarcoma (Berkenblit et al., 2007). In a study on breast cancer, it was reported that ES caused cell death through apoptosis (Qu et al., 2010). Recently, ES has also been used to treat Menkes disease, which is a neurological disorder that happens because of copper deficiency (Guthrie et al., 2020). Based on the fundamental differences in copper metabolism between cancer and normal cells—particularly the elevated copper levels and copper-dependent pro-oncogenic mechanisms in malignancies—ES has emerged as a promising multifunctional drug candidate. It effectively kills cancer cells while also protecting neuronal cells (Gao et al., 2021). Normal cells possess robust copper homeostatic regulation (Ge et al., 2022), intact mitochondrial function, and a potent antioxidant defense system that maintains ROS at low levels (Nagai et al., 2012). As a result, ES-Cu complexes are rarely activated in normal cells, minimizing oxidative stress and preventing cellular damage. So, boosting the level of ES in tumor tissues helps get more copper ions into cancer cells, which leads to the death of those cells (Nagai et al., 2012).

Fe-S clusters are cofactors made up of iron and inorganic sulfur, which play a crucial role in a variety of biological processes, including mitochondrial energy production, amino acid synthesis, cofactor synthesis, and tRNA modification (Lill, 2009). The synthesis of Fe-S clusters involves four steps: 1) Nitrogen fixation 1 homolog (NFS1) catalyzes the conversion of cysteine to alanine, producing persulfide intermediates in the process; 2) The persulfide intermediate binds to iron, forming a [2Fe-2S] cluster on the scaffold protein of the iron-sulfur cluster assembly enzyme (ISCU); 3) The [2Fe-2S] cluster is released from ISCU with the assistance of a partner protein. Some of these clusters are directly inserted into [2Fe-2S] type iron-sulfur proteins, while others go on to form [4Fe-4S] clusters with the cooperation of iron-sulfur cluster assembly 1(ISCA1) and iron-sulfur cluster assembly 2(ISCA2); 4) The ultimately generated [4Fe-4S] clusters are transferred to the [4Fe-4S] type lipoprotein Fe-S protein for maturation (Gourdoupis et al., 2018; Lill and Freibert, 2020; Weiler et al., 2020) (Figure 2).

Excessive copper can compete with iron for metal-binding sites in Fe-S cluster assembly proteins, thereby hindering the biosynthesis of Fe-S clusters in Escherichia coli under both aerobic and anaerobic conditions (Tan et al., 2014). In addition, copper has been shown to impair the assembly of [4Fe-4S] clusters in vitro (Brancaccio et al., 2017). In vivo, too much copper inhibits Fe-S clusters from forming, which reduces Fe-S enzyme activity and harms mitochondrial function (Lill and Freibert, 2020). Mechanistic studies show that human ISCA1, ISCA2, and ISCU bind copper strongly, disrupting the assembly of iron-sulfur clusters (Du et al., 2023). Copper overload reduces overall Fe-S cluster production in cells, leading to functional loss of Fe-S proteins and disruption of mitochondrial homeostasis. In contrast, non-Fe-S proteins remain largely unaffected, indicating the specificity of this process. In human cells, a shortage of Fe-S clusters causes Fe-S apoproteins to fail to mature properly (Gao et al., 2021). These immature proteins are recognized as misfolded or unstable and are targeted for degradation via mechanisms such as the ubiquitin-proteasome system (Maio and Rouault, 2020). This process prevents toxic protein aggregation and helps maintain intracellular stability. When Fe-S cluster biosynthesis is disrupted, lipoic acid synthetase (LIAS) levels drop, inhibiting protein lipidation (Pan et al., 2018; Wang et al., 2017). Collectively, these findings indicate that Fe-S cluster assembly and the functionality of their downstream proteins represent a key molecular target of copper toxicity.

At the subcellular level, mitochondria are highly sensitive to copper-induced cytotoxicity (Lucena-Valera et al., 2023). Cells that rely on mitochondrial respiration are more sensitive to copper ionophores. When mitochondrial function inhibitors and mitochondrial antioxidants were used, they significantly impacted the cells’ sensitivity to copper ionophores. However, mitochondrial oxidative phosphorylation decoupling agents had no effect on copper toxicity, indicating that mitochondrial respiration is crucial in the process of cuproptosis. For example, in ES-sensitive human lung adenocarcinoma (ABC-1) cells, time-dependent dysregulation was observed in multiple TCA cycle-related metabolites, indicating copper’s direct disruption of energy metabolism (Tsvetkov et al., 2022).

FDX1 serves as a key regulator in this process, encoding components of the sulfolipid metabolic pathway and directly modulating protein lipoylation (Rowland et al., 2018).

During lipoyl synthesis, LIAS forms transient covalent bonds with lipoyl carrier proteins and interacts with enzymes including GCSH and lipoyltransferase (LIPT1) (Gourdoupis et al., 2018; Wang et al., 2017; Seidel et al., 2013; Choi et al., 2019). It’s been shown that LIAS function relies on the normal lipidation of Fe-S cluster proteins. When Fe-S cluster biosynthesis is disrupted, LIAS levels drop, inhibiting protein lipoylation (Pan et al., 2018; Wang et al., 2017). FDX1 functions upstream of LIAS and regulates lipoylation by promoting Fe-S cluster assembly and maintaining LIAS stability (Gourdoupis et al., 2018). It directly binds to LIAS and supplies electronic support for S-adenosylmethionine (SAM)-dependent lipoylation reactions. In the presence of ES, large amounts of Cu²⁺ enter mitochondria. FDX1 is a direct target of the ES-Cu²⁺ complex, which binds to and inhibits FDX1 function, resulting in mitochondrial dysfunction (Ni et al., 2019). Additionally, FDX1 acts as a mitochondrial reductase that reduces Cu²⁺ in the ES-Cu²⁺ complex to Cu⁺ (Zheng et al., 2022). The resulting Cu⁺ induces destabilization and loss of Fe-S cluster proteins, which disrupts LIAS synthesis and inhibits protein lipoylation, ultimately blocking the TCA cycle; binding of lipoylated proteins to Cu⁺ triggers protein oligomerization, resulting in proteotoxic stress and ultimately inducing cell death (Tsvetkov et al., 2022) (Figure 3).

Genetic studies demonstrated that deletion of FDX1 or LIAS inhibits copper-induced cell death. Furthermore, FDX1 knockout enhanced cellular resistance to the copper ionophore ES, but did not significantly affect apoptosis inducers or ferroptosis agents (Tsvetkov et al., 2022). The cytotoxic activity of most copper ion-sensitive compounds (e.g., ES) was markedly reduced in cells cultured under glycolytic conditions, indicating a close link between copper metabolism and mitochondrial-mediated protein lipoylation processes (Tsvetkov et al., 2022). Studies have found that knockdown of FDX1 or lipoylation-related enzymes effectively protects cells from copper-induced toxicity (Tsvetkov et al., 2022). Metabolite analysis showed that FDX1 deficiency impairs protein lipoylation by inhibiting pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KGDH) activity in the TCA cycle. This disruption leads to depletion of succinate and accumulation of both pyruvate and α-ketoglutarate (Ni et al., 2019). Knockdown of FDX1 not only completely blocked protein lipoylation but also significantly suppressed cellular respiration, an effect consistent with LIAS deficiency. Additionally, FDX1 knockdown resulted in elevated levels of SAM, an essential substrate for LIAS in the lipoic acid synthesis pathway. These findings suggest that FDX1 functions as an upstream regulator of protein lipoylation by controlling the function of LIAS (Tsvetkov et al., 2022). Lipoylated proteins are essential for copper binding (Tsvetkov et al., 2022).

When copper binds to lipoylated proteins, it causes the proteins to lose their function. Treatment with a copper ionophore can partly fix these functional defects. Additionally, copper binding to lipoylated proteins in the TCA cycle markedly enhances their toxicity. Native gel electrophoresis showed that DLAT’s oligomerization depends on its lipoylation state. Mass spectrometry analysis revealed that copper ionophore treatment causes Fe-S cluster proteins to degrade in an FDX1-dependent way and triggers protein toxicity stress (Tsvetkov et al., 2022). These findings align with previous observations in bacteria and yeast, where copper was found to destabilize proteins containing Fe-S clusters (Brancaccio et al., 2017; Macomber and Imlay, 2009; Chillappagari et al., 2010).

Copper ionophores disrupt the balance of intracellular copper levels, affecting regulatory mechanisms like the copper import gene solute carrier family 31(SLC31A1) and the copper export genes ATP7A and ATP7B. These genes are closely associated with mutations in the copper metabolism-related diseases Menkes disease and Wilson’s disease, respectively (Lutsenko, 2010; Nevitt et al., 2012). CTR1 serves as the primary channel for copper uptake into cells (Lee et al., 2002). ATP7A functions as a steady-state copper exporter, regulating intracellular copper levels to shield cells from copper toxicity (Aubert et al., 2020). ATP7A is highly expressed in enterocytes and plays a crucial role in both copper uptake and excretion processes (Du et al., 2023). Furthermore, ATP7A has the capability to supply copper to various copper-dependent enzymes, thereby influencing numerous facets of tumorigenesis, such as cell proliferation, metastasis, and angiogenesis (Denoyer et al., 2015). Research has demonstrated that the expression of ATP7A in cancerous tissues is much higher than in the adjacent tissues (Li et al., 2019).

A shared mechanism underlies both copper-induced cell death and dysregulation of copper homeostasis. In human embryonic kidney 293 cells expressing the SV40 large T antigen (HEK293T) and ABC1 cells, when SLC31A1 is overexpressed, the cells’ sensitivity to physiological copper concentrations rises significantly. Elevated copper concentrations result in a reduction in the total quantity of proteins related to mitochondrial respiration, a decrease in protein lipidation, a decline in Fe-S cluster proteins, and an upregulation of heat shock protein 70(HSP70) levels (Tsvetkov et al., 2022). However, inhibitors of ferroptosis, necrosis, and apoptosis have proven ineffective in preventing copper-induced cell death. Conversely, copper chelators with bathocuproine disulfonic acid (BCS) and tetrathiomolybdate (TTM), as well as genetic knockout (KO) of FDX1 and LIAS, have all shown the ability to partially rescue cells from copper-induced death (Tsvetkov et al., 2022). Loss-of-function experiments have further shown that silencing ATP7A can markedly inhibit the growth and metastasis of breast and lung cancer cells (Shanbhag et al., 2019). These findings underscore the pivotal role of copper homeostasis mechanisms in cell survival, tumorigenesis, and sensitivity to copper toxicity. They also suggest that genes involved in copper homeostasis (e.g., SLC31A1, ATP7A) may represent potential therapeutic targets.

Tumor Protein p53 (TP53) is the gene most frequently mutated in human cancers, and the majority of these cancer-related protein 53 (p53) mutations are missense mutations, typically found in its DNA-binding domains. Many of these mutant p53 (mtp53) proteins promote cancer development by gaining new, harmful functions, known as “gain-of-function” (GOF) activity (Mantovani et al., 2019; Zhang et al., 2020). Studies have demonstrated that various mtp53 proteins can play distinct roles in regulating glycolysis and mitochondrial metabolism (Eriksson et al., 2017). The p53 protein, encoded by the TP53 gene, is a key transcription factor activated by diverse cellular stress signals—including DNA damage (Fagherazzi et al., 2025), hypoxia (Simon-Molas et al., 2021), and oncogene activation—to execute central regulatory functions (Gong et al., 2016). Activated p53 transcriptionally regulates downstream target genes (e.g., the cell cycle inhibitor p21)to arrest the cell cycle at the G1/S checkpoint, providing time for DNA repair. If the damage is irreparable, p53 initiates apoptosis to eliminate abnormal cells (Liu et al., 2024; Baliakas and Soussi, 2025). Additionally, p53 plays a broad role in regulating cellular metabolism, influencing key processes such as glycolysis, oxidative phosphorylation, and antioxidant responses (Sanford et al., 2023). It also induces cellular senescence and irreversible cell cycle arrest, thereby suppressing tumor progression (Liu et al., 2024). p53 also plays an important role in maintaining intracellular copper homeostasis through two primary mechanisms:

On one hand, p53 inhibits the copper chaperone ATOX1 to reduce free intracellular copper availability and prevents excess copper accumulation (Tsymbal et al., 2023). It also indirectly senses copper levels by modulating glutathione (GSH) concentrations (Xiong et al., 2023). On the other hand, under pathological or experimental conditions with abnormally high copper, p53 eliminates copper-damaged cells to protect the organism (Xiong et al., 2023).

Possible mechanisms of action of p53 in copper-induced cell death, also known as “cuproptosis,” include:

1. Promote cuproptosis sensitivity: p53 may enhance cells’ sensitivity to cuproptosis by suppressing glycolysis and facilitating the shift towards mitochondrial metabolism. Firstly, p53 reduces glucose uptake by suppressing the expression and activity of glucose transporters such as glucose transporter 1 (GLUT1) and glucose transporter 4 (GLUT4) (Kawauchi et al., 2008; Schwartzenberg-Bar-Yoseph et al., 2004). Secondly,p53 suppresses glycolysis by inhibiting several key catalytic steps within the glycolytic pathway. For example, p53 inhibits the conversion of glucose to glucose-6-phosphate by promoting the degradation of Hexokinase 2(HK2) mRNA (Wang et al., 2014). In the final step of glycolysis, pyruvate is converted to lactate by lactate dehydrogenase (LDHA/B). p53 represses LDHA expression either by directly binding to its promoter or by promoting degradation of the LDHA transcription factor HIF-1α (Zhou et al., 2019; Semenza, 2010). This can result in the accumulation of intracellular pyruvate, which subsequently enters the TCA cycle and supports oxidative phosphorylation.

In summary, a necessary condition for the occurrence of copper-induced cell death is that the concentration of copper ions exceeds normal levels. When copper overload happens, copper competes with iron for the metal-binding sites in Fe-S cluster assembly proteins. This competition interferes with the formation of Fe-S cluster proteins. This leads to a decrease in LIAS levels and inhibits the acylation of proteins. However, in the presence of FDX1, LIAS can be stably generated and directly binds to FDX1. This interaction promotes the lipoylation of proteins. The lipoylated proteins then bind to Cu⁺, which initiates protein oligomerization. This oligomerization leads to protein toxic stress, ultimately inducing cell death. Overexpression of genes involved in copper homeostasis can enhance a cell’s sensitivity to copper concentrations. Additionally, p53 increases cellular sensitivity to cuproptosis by suppressing glycolysis and maintaining normal mitochondrial function.