Harnessing copper: Innovative approaches to combat neurodegenerative diseases and cancer (Review).

1.
Introduction
Copper is an essential trace element and the recommended daily intake for adults ranges from 0.8 to 2.4 mg per day (1). Copper is required for almost all the functions of tissues and organs, in particular for oxidative phosphorylation in the heart and brain, scavenging of free radicals (2), angiogenesis (3), bone formation and regeneration (4,5), modulation of bone strength (6,7) and the regulation of rest-activity cycles (8) (Fig. 1). Copper also participates in the synthesis of copper-dependent enzymes, including superoxide dismutase (SOD) (9), cytochrome c oxidase (10), tyrosinase (11), lysyl oxidase and dopamine (DA) β-hydroxylase (12,13). Copper enzymes play a crucial role in key physiological processes such as energy metabolism, antioxidant defense and neurotransmitter synthesis. Their dysfunction directly leads to a variety of genetic and acquired diseases (14,15) (Table I).
Copper homeostasis regulation can be classified into three processes: Absorption, utilization/storage and excretion. Disruptions in copper homeostasis can result in impaired physiological functions and the onset of various related diseases (Fig. 1). In response to these challenges, researchers have developed a range of copper-targeted therapeutic strategies designed to manage such conditions. These interventions specifically address pathological states caused by copper deficiency, copper overload or disturbances in copper metabolism, and they exert their effects by modulating copper concentration, distribution or biological activity within the body. These therapeutic approaches show significant potential in the treatment of hereditary copper metabolism disorders, neurodegenerative diseases and cancers. This article provides a comprehensive and timely review of the dual role of copper in human biology, with a particular emphasis on its implications for health, neurodegenerative diseases and cancer. This review provides a theoretical foundation and offers diverse perspectives for the development of treatments for related diseases.

2.
Maintenance of copper homeostasis

Absorption and transport of copper
Copper absorption primarily occurs in the small intestine, particularly in the duodenum, which serves as the key site for this process (16). In dietary sources, copper is predominantly present as Cu(II). The six-transmembrane epithelial antigen of the prostate proteins (17), localized on the apical membrane of small intestinal epithelial cells, possess reductase activity that facilitates the reduction of Cu(II) to Cu(I). Subsequently, Cu(I) is taken up by the copper transporter 1 (CTR1), also known as solute carrier family 31 member 1 (SLC31A1) (18,19), which is expressed on the intestinal epithelial membrane. Once Cu(I) enters the enterocytes, it is transported into the bloodstream through vesicular transport mediated by ATPase copper transporting α (ATP7A) (Fig. 2). In the bloodstream, copper binds to various proteins, including human serum albumin (20), ceruloplasmin (21), albumin, α-2-macroglobulin (22), histidine and transcupreins. These complexes either support copper's physiological functions through the systemic circulation or enable its uptake and storage by the liver (23-25).
The high-affinity copper transporter protein CTR1 facilitates the transfer of extracellular Cu(I) into the cell (26). In mice, intestinal-specific knockout of CTR1 resulted in severe growth and developmental defects, leading to embryonic lethality by mid-gestation. These findings indicate that CTR1-mediated copper uptake is essential for mammalian copper homeostasis and embryonic development (27). Additionally, CTR1 gene expression is regulated by changes in specificity protein 1 (Sp1) activity and extracellular copper concentrations (28,29). Specifically, the human high-affinity copper transporter hCTR1 is transcriptionally upregulated during copper deficiency and downregulated under copper-replete conditions. Elevated hCTR1 levels also suppress its own expression. Notably, Sp1 modulates hCTR1 expression under copper stress (30).

Copper utilization and storage in cells
Once inside the cell, copper rapidly binds to co-chaperone proteins such as antioxidant 1 copper chaperone (Atox1), copper chaperone for SOD (CCS) and cytochrome c oxidase copper chaperone COX17 (COX17). This interaction is essential for the safe and targeted delivery of copper to its specific functional sites, thereby supporting and maintaining critical cellular processes (29).
Atox1 functions as a key cytoplasmic copper chaperone, directly binding Cu(I) and mediating its delivery to the copper-transporting ATPases ATP7A and ATP7B located on the Golgi membrane (Fig. 2). This process is essential for the systemic distribution of copper ions and the maintenance of copper homeostasis (31,32). Importantly, research by Lutsenko has revealed that Atox1 promotes the synthesis of ceruloplasmin, a protein critical for iron metabolism, as well as tyrosinases (33).
The CCS is localized in compartments such as the cytoplasm and the mitochondrial intermembrane space (34), binds copper and activates SOD1 (Cu/Zn SOD), a copper/zinc-dependent enzyme. In eukaryotes, SOD is expressed as SOD1 in the cytoplasm and extracellularly, and as Mn-SOD2 in the mitochondria. The SOD enzymes catalyze the disproportionation of superoxide (O2•-) to produce hydrogen peroxide (H2O2), and the balance between copper and reactive oxygen species (ROS) is physiologically crucial (35,36). Furthermore, CCS expression is strongly negatively correlated with copper levels, serving as a feedback regulatory mechanism to prevent excessive activation of SOD1 under conditions of copper overload (37).
The COX is associated with oxidative phosphorylation in mitochondria, and its function is mainly mediated by five proteins: COX17, synthesis of cytochrome C oxidase 1 (SCO1), SCO2, cytochrome c oxidase assembly factor 6 (COA6) and COX11, each of which is essential. COX17 transfers copper ions from the cytoplasm to the mitochondrial intermembrane space (38), and then, through the formation of a disulfide bond, transfers them to SCO1 (39). Copper is transported primarily through the action of COA6 with SCO2 in the maintenance of redox homeostasis within mitochondria or through COX11 (40).
In addition to these chaperones, copper can be transported into the mitochondria by the mitochondrial phosphate transporter SLC25A3 (41). Another key component is the intracellular copper storage proteins metallothionein 1 (MT1) and MT2, which have a high affinity for copper (40), while copper, as a catalyst for the Fenton reaction, is also capable of binding to glutathione (GSH) (42). These proteins are essential for the effective utilization of copper and the synthesis of key copper enzymes, including SOD, cytochrome c oxidase, tyrosinase, lysyl oxidase, DA β-hydroxylase, ATPase copper-transporting α (ATP7A) and ATP7B to maintain normal cellular functions (40).

Regulation by intracellular copper levels
The regulation of copper homeostasis is fundamentally dependent on the copper-transporting ATPases ATP7A and ATP7B. In response to elevated intracellular copper [Cu(I)] levels, ATP7A and ATP7B translocate from the trans-Golgi network (TGN) to vesicles that subsequently fuse with the plasma membrane, thereby facilitating the efflux of Cu(I) from the cell. Conversely, when intracellular Cu(I) levels are reduced, these ATPases are retained within the TGN, where they actively transport Cu(I) into the TGN lumen to support the maturation of copper-dependent proteins. Once physiological Cu(I) levels are restored, ATP7A and ATP7B are recycled back to the TGN, re-establishing their basal localization and maintaining responsiveness to fluctuations in cellular copper concentrations (43). ATP7A can mediate copper transport across polarized cellular barriers, such as the blood-brain barrier (BBB) and the blood-placenta barrier, thereby enabling copper export and ensuring that the fetus receives adequate copper during development. ATP7B, predominantly expressed in the liver, becomes active under conditions of elevated hepatic copper concentrations (33). In hepatic cells, ATP7B plays a crucial role in the regulation of copper homeostasis by mediating the transport of excess copper into bile, leading to its excretion via feces, a process essential for maintaining the systemic copper balance (44). This protein facilitates copper elimination via bile secretion or through binding to soluble proteins for delivery to specific tissues and organs (45).

3.
Impact of copper on health

Copper deficiency
Copper deficiency is associated with impaired immune function and reduced T-cell proliferation, which leads to decreased production of interleukin-2 (IL-2). The capacity of immune cells to generate superoxide anions and eliminate bacteria is diminished in mild copper deficiency, and the number of neutrophils in the peripheral blood is reduced in severe copper deficiency (46,47). Menkes syndrome (MD) is an X-linked recessive disorder caused by mutations in ATP7A and is characterized by systemic copper deficiency, primarily due to impaired intestinal copper uptake, progressive neurodegeneration, connective tissue disorders, and brittle, kinky hair (48). Infants with severe MD often do not survive beyond the third year of life, with neurological deficits, matted hair, developmental delays, genitourinary abnormalities, skin abnormalities, vascular abnormalities, skeletal abnormalities and spasticity of the limbs that transform into muscle weakness of the limbs along with other abnormal signs. Most patients die within the third year of life from cerebral hemorrhage due to vascular fragility or infection-related complications (49). This indicates that copper plays a crucial role in infant development: Copper imbalance driven by ATP7A mutations leads to extensive multi-organ dysfunction in MD, which is manifested as multi-system abnormalities and fatal outcomes in infants. Another disorder caused by mutations in ATP7A leading to copper deficiency is occipital horn syndrome (OHS), which is a phenotype of MD but with milder symptoms than those of MD (50). Bone exostoses, radial head dislocations, keloid-like skin lesions and dental abnormalities are specific to OHS, which can also present with developmental delay and mild neurological symptoms (14). An early study indicated that the ATP7A transcript lacking exon 10 encodes a partially functional protein, and this residual function is associated with a milder form of occipital horn syndrome (OHS), which is caused by mutations at the ATP7A splicing site. Unlike the loss-of-function mutations observed in classical Menkes disease (MD), OHS is characterized by relatively mild clinical symptoms (50). In 2022, Batzios et al (51) identified a genetic disorder of brain copper metabolism caused by CTR1 deficiency, which presents with hypotonia, global developmental delay, seizures and rapid cerebral atrophy. Brain CTR1, ATP7A and ATP7B protein levels may be affected by factors other than gene expression, such as the rate of copper transport mediated by these proteins. The key to treating MD is supplementation with additional copper (52-54), which is not replenished orally because of impaired intestinal absorption caused by ATP7A deficiency. The most widely used treatment is parenteral or subcutaneous supplementation with copper histidine (55,56).

Copper overload
Copper overload disrupts the expression and function of antioxidant enzymes and induces oxidative stress, while oxidative stress and increased ROS levels are thought to be the main cause of Cu-induced cytotoxicity (57,58). When copper is overloaded, hydrogen peroxide can be converted into highly reactive hydroxyl radicals, which can cause DNA and membrane damage (59,60). Excessive copper intake has also been linked to alterations in the gut microbiota, which in turn can lead to a range of conditions such as polycystic ovary syndrome (61). Studies have shown that increased intracellular Cu activates autophagy through unc-51 like autophagy activating kinase 1 (ULK1)/ULK2 signaling, thereby promoting cancer cell growth and survival (62,63). Wilson disease (WD) is an autosomal recessive disorder caused by mutations in ATP7B that is primarily due to ATP7B mutations that result in impaired copper excretion from hepatocytes into bile, a significant increase in copper levels in hepatocytes and subsequent liver injury; excess copper is released into the circulation, where it is deposited in areas such as the brain or the eyeballs, causing damage. WD is clinically heterogeneous and may present with neurological dysfunction, acute liver failure and the presence of hemolysis, rhabdomyolysis, renal tubular injury, leukopenia and thrombocytopenia (64-66). Long-term management of WD includes chelating agents such as tetrathiomolybdate (TM) (67), trientine or d-penicillamine (68-70), as well as zinc salts to reduce copper absorption (40,71,72).

4.
Neurodegenerative diseases

Alzheimer's disease (AD)
AD is a neurodegenerative disorder characterized by the presence of β-amyloid (Aβ) plaques and tau protein tangles in the brain (Fig. 3A). Initially, these pathological features were regarded as the primary pathogenic drivers of AD, prompting a focus on therapeutic strategies aimed at reducing the accumulation of Aβ or tau proteins (73,74). However, drug development targeting these proteins has faced substantial challenges: >30 Phase III clinical trials have shown that Aβ inhibitors do not significantly improve cognitive function in patients with AD, and some interventions have even led to adverse effects following plaque removal (75-77). This research and development predicament not only highlights the limitations of traditional target therapy but also prompts researchers to re-examine and explore the pathogenesis of AD.
While the precise molecular mechanisms underlying AD remain elusive, accumulating evidence suggests that dysregulation of brain metal ions, particularly copper and zinc, may contribute to the hallmark Aβ neuropathology associated with the disease (78,79). Transition metals such as copper, iron and zinc are essential for normal physiological functions; however, copper has been specifically implicated in promoting Aβ aggregation and the formation of harmful species. Notably, individuals with AD exhibit reduced copper levels in brain tissue, yet elevated concentrations in serum and age-related pigments (80-82). Copper's role in promoting Aβ precipitation and generating redox-active species warrants further investigation. Serum levels of both free and total copper are significantly elevated in individuals with AD, and increased copper deposition is observed in the age-related pigments of these patients (82,83). Following the observation that copper promotes Aβ precipitation under acidic conditions, the interactions between copper and Aβ have been increasingly explored in detail (84-86). Critically, copper can form complexes with Aβ that drive redox reactions, involving lipids to induce oxidative stress, ultimately leading to cellular apoptosis and contributing to cognitive decline (87-89).

Research on the treatment of AD with copper
In early animal studies, inhibition of CTR1 in Drosophila was found to improve AD-like symptoms caused by Aβ42 (90). In the preventive paradigm of the Tg2576 mouse model of AD, TM treatment reduced brain copper levels and lowered Aβ concentrations (91,92). Excessive circulating copper may impair brain function in wild-type mice and worsen neurodegenerative changes in AD mouse models (93,94). Another study demonstrated that long-term oral exposure to very high levels of copper or zinc resulted in only minimal alterations in brain metal homeostasis. Specifically, in transgenic APP-C100 mice (engineered to express a mutant form of the human amyloid precursor protein without the typical Aβ deposition characteristic of AD), high-level metal exposure does not induce Aβ protein formation (95). These findings suggest that modulating copper transport or levels - such as through CTR1 inhibition and the use of copper regulators - may alleviate Aβ-related AD pathology. However, excessive copper exposure can worsen the condition, and therapeutic efficacy may depend on the presence of Aβ deposition, as copper shows little effect in models without amyloid deposits. In the brain, high extracellular copper levels promote amyloid formation, and low intracellular copper levels also lead to amyloid formation (96-98), suggesting that the symptoms of AD are complex and difficult to treat with copper chelators or copper ion carriers alone (99,100). Therefore, enhancing copper transport to the brain to increase its bioavailability has become a key strategy for the treatment of AD (101), for instance, with quinolones (clioquinol and PBT2) or bis(thiosemicarbazones) (BTSCs) (102-104). BTSCs restore cognitive function by increasing copper bioavailability and inhibiting the formation of neurotoxic Aβ trimers and phosphorylated Tau (105), whereas clioquinol and PBT2 act as Zn2+-Cu2+ ion carriers (106,107), regulating the intracellular concentration and distribution of zinc and copper ions, leading to the reduction of Aβ along with high intracellular levels of copper and zinc (73,108). The mechanism of action of PBT2 has also been investigated, but the results of the clinical trials (no. ACTRN12611001008910) for AD therapy have not been satisfactory, as Pittsburgh compound-B standardized uptake value ratios in the placebo group exhibited a surprising decline, obscuring statistically significant differences compared to the treatment group and even showing a more pronounced reduction. Recently, it was concluded that Aβ peptides associated with AD form a unique Cu(Aβ) complex to which PBT2 cannot effectively bind. Further study has shown that the chelator PBT2 forms a ternary Cu(II) complex with Aβ that is highly stable but has low specificity (109). This remains a major challenge in the treatment of AD with copper.

Huntington's disease (HD)
HD is an inherited neurodegenerative disorder characterized by neuropsychiatric symptoms, movement disorders (most commonly choreoathetosis) and progressive cognitive impairment. HD is currently treated symptomatically and scientists have struggled to identify effective disease-modifying therapies. Metal homeostasis is disrupted during the progression of HD, although its exact role in the pathogenesis of the disease remains elusive. Meanwhile, some researchers have observed tissue abnormalities and deposition of copper (Cu) and zinc (Zn) in HD-affected brain regions, which may contribute to disease initiation and progression through mechanisms including mitochondrial dysfunction, oxidative stress and BBB dysfunction (110). It has been reported that copper-regulated genes are upregulated in HD and that copper binds to the N-terminal fragment of huntingtin, supporting the involvement of aberrant copper metabolism in HD. Copper has been shown to accelerate fibril formation of the N-terminal fragment of huntingtin in vitro via the expanded polyglutamine (polyQ) stretch (httExon1). Copper has also been found to enhance polyQ aggregation and toxicity in mammalian cells expressing httExon1. Studies in a yeast model of HD demonstrated that overexpression of several genes involved in copper metabolism reduced polyQ-mediated toxicity. The MT3 gene belongs to the metallothionein family and is mainly expressed in the central nervous system. It regulates zinc and copper homeostasis and acts as a neuronal growth inhibitory factor (111). Overexpression of MT3 in mammalian cells significantly reduced polyQ aggregation and toxicity (112). Animal studies have also shown that copper chelators modulate the early events of Htt misfolding and reduce neurotoxicity in the Drosophila HD model (90,113,114). Despite the promising therapeutic potential of MT3 in HD, its clinical translation remains limited by technical and mechanistic challenges.

Parkinson's disease (PD)
PD is an age-related neurodegenerative disease, and the role of oxidative stress and mitochondrial dysfunction in the progression of PD has been widely recognized in relation to the selective loss of DA neurons in the substantia nigra pars compacta (SNpc) of the nigrostriatal DA pathway and the reduction of DA levels in the striatum (115). The normal substantia nigra contains twice as much copper as other brain regions, suggesting that copper plays an important role in this brain area (116). Autopsy brain samples from patients with PD show increased iron and decreased copper in the substantia nigra and basal ganglia (117,118).
At the cellular level, PD is associated with the overproduction of ROS. While copper may contribute to intracellular oxidative stress by participating in the Fenton and Haber-Weiss reactions or by interfering with iron homeostasis, either directly or indirectly through the formation of hydroxyl radicals, experimental evidence linking copper deficiency and the formation of SOD1 aggregates to the progression of PD is also discussed in terms of its therapeutic implications (119,120). Copper causes a decrease in SOD1 activity by interfering with SOD1 synthesis, leading to a loss of cellular protection against neuronal oxidative damage (121,122).
It is important to note that mitochondrial dysfunction represents a central mechanism driving oxidative stress in PD. Mutations in the phosphatase and tensin homolog-induced kinase 1 (PINK1) and PARKIN RBR E3 ubiquitin ligase (PARKIN) genes are associated with familial forms of the disease. Parkin and PINK1 play critical roles in mitochondrial quality control, particularly in the process of mitophagy, and are essential for the survival of dopaminergic neurons. Under normal physiological conditions, PINK1 accumulates on damaged mitochondria by sensing changes in the mitochondrial membrane potential. This accumulation promotes Parkin recruitment and activates its E3 ubiquitin ligase activity, resulting in the ubiquitination of damaged mitochondria and the initiation of autophagic degradation. This pathway limits the release of ROS and reduces mitochondrial toxicity (123,124).
Dysregulation of copper homeostasis interferes with the PARKIN/PINK1 pathway through multi-layered mechanisms (125). Excessive copper downregulates the transcription of PINK1 and PARKIN in cellular models, although the exact regulatory mechanism (e.g., involvement of NF-κB) remains to be fully elucidated. Importantly, copper can also impair the activity of PINK1 and PARKIN proteins by inducing conformational changes, specifically through binding to the kinase domain of PINK1 and the RING finger domain of PARKIN. Notably, the role of copper in regulating PINK1/PARKIN is context-dependent: It inhibits PINK1/PARKIN function under pathological conditions (e.g., copper excess), while promoting their transcriptional regulation and autophagic processes under physiological conditions (125,126). It further impairs PINK1 kinase activity and Parkin E3 ubiquitin ligase activity by coordinating with cysteine/histidine residues within their respective functional domains, the kinase domain of PINK1 and the RING finger domain of Parkin, thereby inducing conformational changes (125). These disruptions prevent Parkin recruitment to damaged mitochondria, inhibit mitochondrial substrate ubiquitination and perturb the PARKIN/PINK1-regulated balance of mitochondrial dynamics, ultimately leading to the accumulation of dysfunctional mitochondria and exacerbated oxidative stress. This cascade reflects the pathological shift in copper's regulatory role in mitophagy under homeostatic imbalance, which contrasts with its physiological role in promoting mitophagy-mediated tissue regeneration (126). However, the underlying mechanisms remain incompletely understood and require further investigation. In addition, exposure to elevated copper levels has been shown to accelerate the formation of toxic α-synuclein aggregates, a key pathological contributor to neuronal loss in PD (127).
Second, microglia-mediated neuroinflammation is an important component of the pathogenesis of PD. In animal studies, researchers have observed inflammatory changes in mouse brain tissue, including activation of microglia, loss of dopaminergic neurons and aggregation of α-syn in the substantia nigra. Copper has been shown to activate BV2 cells via the NF-κB pathway and to increase ROS levels in BV2 cells (128,129). Sustained copper accumulation in BV2 cells resulted in a decrease in mitochondrial membrane potential, a reduction in Parkin and PINK1 expression, an increase in P62 expression and light chain 3BII/I ratio, and upregulation of NLR family pyrin domain containing 3/caspase-1/gasdermin D axis proteins (130,131).
Research on the treatment of PD with copper-based compounds, such as 8-hydroxyquinoline-2-carboxaldehyde isonicotinoylhydrazine, a moderate metal-binding compound that binds to copper ions, has been shown to efficiently compete with ortho-nucleosides for Cu(I) and Cu(II) binding and to inhibit protein aggregation in vitro and in cells (132). Copper(II) diacetylbis(4-methylthiosemicarbazone), also known as CuII(atsm), is an orally available, biologically active BBB-penetrant compound that is widely used to selectively label hypoxic tissues in cellular imaging experiments (133). It has been shown to accumulate in the striatum of patients with PD at levels that positively correlate with disease stage (134). Furthermore, CuII(atsm) has demonstrated therapeutic potential in rescuing dopaminergic neuron loss and alleviating motor dysfunction in PD models (119,135). In addition, CuII(atsm) supplementation has been shown to significantly reduce the misfolding and deposition of wild-type SOD1 protein associated with PD in a novel mouse model, enhance DA neuron survival and improve motor function, suggesting its potential as a novel therapeutic strategy for PD treatment (136). However, further research and clinical trials are necessary to validate these findings.

Amyotrophic lateral sclerosis (ALS)
ALS is a progressive neurodegenerative disease of the motor neurons that leads to the worsening of casual muscle weakness until death from respiratory failure ~3 years after onset (137). Numerous symptoms of ALS are associated with signs of copper deficiency, leading to defects in the vasculature, antioxidant system and mitochondrial oxidative respiration, but there are also signs of copper toxicity, such as increased ROS generation and protein aggregation (138).
About 2% of ALS cases have mutations in SOD1, which neutralizes harmful reactive oxygen superoxide (139). Familial ALS is at times linked to the gene encoding Cu/Zn-binding SOD. Mutations in ALS are thought to result in functional enhancement of dismutase activity (140). Mutations in human SOD1 are found in ~20% of patients with familial ALS. They are characterized by a conformational dysfunction of the electrostatic and zinc-binding elements (mutant SOD1-mediated pathogenesis of ALS). A study using transgenic mice expressing wild-type or mutant human SOD confirmed that dominant mutations in SOD and the acquisition of function are important factors in the pathogenesis of familial ALS (141).
Previous studies have shown that CuII(atsm) in ALS can restore SOD1 dysfunction caused by copper deficiency (135). Therefore, the use of Cu as a new target for treating familial progressive ALS becomes possible. However, further studies are necessary to validate these findings.
Overall, for copper-related diseases, including both the copper metabolism disorders discussed earlier and the neurodegenerative conditions addressed in this chapter, therapeutic strategies targeting copper homeostasis have been developed, with drug selection guided by the underlying disease etiology (deficiency, overload or imbalance). This establishes a clear pathophysiological and etiological foundation for clinical intervention (Table II).

5.
Copper and cancer

Epidemiological context of cancer incidence
According to the most recent estimates provided by the International Agency for Research on Cancer, ~20 million new cancer cases, including non-melanoma skin cancers, and 9.7 million cancer-related deaths were reported globally in 2022. Lung cancer emerged as the most frequently diagnosed cancer, accounting for nearly 2.5 million new cases, representing approximately one in eight cancer diagnoses worldwide (12.4% of all cancers). This was followed by female breast cancer (11.6%), colorectal cancer (9.6%), prostate cancer (7.3%) and gastric cancer (4.9%). Furthermore, lung cancer was identified as the leading cause of cancer-related mortality, with an estimated 1.8 million deaths (18.7% of total cancer deaths), followed by colorectal cancer (9.3%), liver cancer (7.8%), female breast cancer (6.9%) and stomach cancer (6.8%) (142).
Abnormal accumulation or deficiency of trace elements plays a significant role in cancer pathogenesis and progression, with dysregulation of copper metabolism being particularly notable (143,144). Research suggests that there is an increased demand for copper during tumor proliferation and metastasis (40,145). Elevated serum copper levels have been reported in various cancers, including breast cancer (146,147), lung cancer (148) and prostate cancer (149). By contrast, reduced serum copper levels have been observed specifically in hepatocellular carcinoma and endometrial cancer (150,151), although the underlying mechanisms remain to be fully elucidated.

Molecular mechanisms of copper in promoting carcinogenesis
Copper can promote tumorigenesis by promoting oxidative stress that damages DNA and related molecular structures (152), and contributes to tumor development by promoting angiogenesis, metastasis and cell proliferation through multiple mechanisms: Increased intracellular copper enhances oxidative stress caused by the formation of ROS, leading to elevated intracellular ROS levels (153), DNA damage and activation of oncogenes (59), whereas reduced intracellular copper results in decreased SOD1 activity, nearly complete loss of resistance to oxidative damage and impaired maintenance of normal cellular life activities (121).
McAuslan and Reilly (154) were the first to discover that copper promotes migration of vascular endothelial cells. Narayanan et al (155) found that angiogenesis is inhibited after silencing CTR1 using small interfering RNAs. Furthermore, copper has been shown to regulate the secretion of vascular growth factors, including fibroblast growth factor (FGF) and IL-1α (156,157), and to modulate endothelial cell affinity by binding to angiogenic factors (158). TM-induced copper deficiency leads to reduced transcriptional activity of NF-κB and suppressed expression of the angiogenic factors FGF (159), VEGF (160), IL-6, IL-8 and IL-1α (161-163); copper activates hypoxia-inducible factor (HIF)-1, and when copper is chelated, HIF-1-mediated VEGF expression is blocked (164,165).
Copper has also been found to directly bind ULK1/ULK2 and activate autophagy by Tsang et al (62). Cellular autophagy is inhibited when CTR1 is deficient, which can suppress oncogene-induced cancer cell proliferation; autophagosomes are reduced and tumor size is significantly decreased in mice in which CTR1 is knocked out using single-guide RNA (62). Copper promotes cancer by stimulating the MAPK pathway (166). Copper binds to MAPK kinase (MEK)1/2 and activates downstream ERK1/2 phosphorylation, thereby promoting tumor proliferation. MEK1/2 activity was inhibited in BRAFV600E-positive cancer cells, and proliferation was significantly suppressed after TM treatment, suggesting that MEK1/2 activity is highly correlated with copper levels (167,168). The epidermal growth factor receptor (EGFR), as a key upstream regulator of the MAPK pathway, is overexpressed or mutated in various cancers (including ovarian cancer), driving cell proliferation, survival and metastasis. Copper binds to and inhibits protein tyrosine phosphatase N2 (PTPN2), leading to the loss of its phosphatase activity. This inhibition relieves the suppression of EGFR dephosphorylation, thereby activating EGFR signaling. In turn, activated EGFR suppresses the transcription of CTR1 via the cAMP responsive element binding protein (CREB) signaling pathway, forming a closed-loop regulatory circuit: 'copper-PTPN2-EGFR-CREB-CTR1'. Fluctuations in intracellular copper levels can thus modulate EGFR activity through this feedback axis, highlighting a mechanistic link between copper homeostasis and EGFR signaling (169). Research by Jakhmola et al (147) revealed the significant role of EGFR in ovarian cancer through computational simulation methods and pointed out that the DADETL segment (including the self-phosphorylated tyrosine 992) is the key region leading to receptor overexpression. This is similar to the copper-mediated activation mechanism of the MAPK pathway, suggesting that copper may indirectly affect tumor development by regulating EGFR activity or downstream signal transduction. Future studies are necessary to deeply explore the direct association between copper and the EGFR pathway, providing new ideas for the combined application of copper-targeted therapy and EGFR inhibitors (147).
Copper also mediates the activation of programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) in cancer cells, enabling immune evasion. When copper chelators are used, phosphorylation of STAT3 and EGFR is inhibited, promoting ubiquitination-mediated degradation of PD-L1, and slowing tumor growth by increasing the number of CD8+ T cells and NK cells (170,171).

Mechanisms and implications of cuproptosis for cancer therapy
It has been shown that the metabolic profile of cancer cells differs from that of normal cells in that cancer cells maintain a higher metabolic rate to support a higher proliferation rate and to resist cell death signals (172). Cell death involves proteins and lipids and includes apoptosis (173,174), necroptosis (175), pyroptosis (176) and ferroptosis (177,178). The demand for copper is high during cell growth and abnormal accumulation of excess copper within the cell leads to cellular dysfunction and ultimately cell death (179,180). Mechanisms of copper-induced toxicity include induction of apoptosis (181-183), caspase-independent cell death pathways (184-186), induction of ROS or inhibition of the ubiquitin-proteasome system (129,187-189).
However, the mechanism underlying copper-induced cell death remained unclear until a study published in Science in 2022 by Tsvetkov et al (190) proposed the concept of cuproptosis - a novel form of copper-induced cell death that is distinct from other known forms of cell death (Fig. 3B). This study demonstrated that copper ionophore-induced cell death is predominantly dependent on the accumulation of intracellular copper (191,192). Notably, treatment with inhibitors targeting other established mechanisms of cell death, including ferroptotic apoptosis (ferrostatin-1), necroptosis (necrostatin-1) and oxidative stress (N-acetylcysteine), did not abolish copper ionophore-induced cytotoxicity (190). Genome-wide CRISPR/Cas9 knockdown experiments identified seven specific genes that significantly mitigated the cytotoxic effects associated with the copper ionophore elesclomol (193). Among the identified genes was ferredoxin 1 (FDX1), a reductase known for its role in reducing Cu(II) to the more toxic Cu(I) and serving as a direct target for elesclomol (194,195). Additionally, six genes encoding components of the lipoic acid pathway - specifically lipoyltransferase 1, lipoic acid synthase (LIAS) and dihydrolipoamide dehydrogenase - or targets associated with lipoylated proteins within the pyruvate dehydrogenase complex [including dihydrolipoyl transacetylase (DLAT), pyruvate dehydrogenase E1 subunit α1 (PDHA1) and PDHB] were also identified (196). Importantly, single gene knockout analyses revealed that FDX1 and LIAS were particularly effective at diminishing cytotoxicity associated with copper ion carriers. Furthermore, inhibition of complex I was found to obstruct copper-induced cell death. This finding suggests that applying an electron transport chain inhibitor can prevent the cytotoxic effects induced by copper when administered alongside elesclomol in both serum-containing and serum-free media. Under serum-free conditions, cells exhibited significant resistance to elesclomol-mediated cytotoxicity; substantial toxicity was observed only when elesclomol-Cu(II) complexes were present. This effect was inhibited by the addition of TM. Furthermore, buthionine sulfoximine has been demonstrated to sensitize cells to copper-induced cell death by inhibiting glutamate cysteine ligase, which results in a direct depletion of the endogenous copper chelator GSH. The underlying mechanism involves the role of FDX1 in promoting the lipoylation of DLAT and dihydrolipoamide S-succinyltransferase proteins (190,197). Cu(II) binds to lipoylated DLAT, facilitating the production of insoluble DLAT that promotes cell death. Conversely, the conversion of Cu(II) to the more toxic Cu(I) induces cell death and intracellular oxidative stress, leading to an increase in heat shock protein 70 expression levels (190).
P53 activity maintains the functional integrity of mitochondrial morphology and structure, and mutations or deficiencies in p53 lead to significant reductions in cytochrome c oxidase (COX) activity and respiratory metabolism (198-200). P53 also promotes the conversion of pyruvate to acetyl coenzyme A by promoting lactate dehydrogenase A to maintain high pyruvate levels and by promoting dephosphorylation to activate PDH complexes (201,202). Copper-induced cell death is also highly dependent on cellular oxidative phosphorylation, the circulating component that targets tricarboxylic acid, and cells are more susceptible to copper death when glycolysis is inhibited. Studies have shown that cells dependent on mitochondrial oxidative phosphorylation for energy are 1,000-fold more sensitive to copper ion carriers than glycolysis-dependent cells (190).

Potential of copper-targeted cancer therapy
New studies have also shown that advanced clear cell renal cell carcinoma (ccRCC) accumulates Cu and allocates it to CuCOX. Copper promotes ccRCC growth through a combination of bioenergetics, biosynthesis and redox oncogenic remodeling of body homeostasis (203).
Through bioinformatics analysis, more and more researchers are focusing on the important link between cuproptosis and the cancer process (128,204). With the discovery of cuproptosis (205-207), the interactions between copper ionophores (208,209), copper and mitochondria, and drugs that had previously been considered as potential anti-tumor therapies (71), have become increasingly clear. This has accelerated research into copper-targeting therapies, with numerous studies on copper chelators and ionophores demonstrating significant therapeutic efficacy across various cancer types (Table III).

6.
Clinical translation and challenges of copper-targeting approaches
Copper-targeted therapies exhibit promising potential in the treatment of cancer and neurodegenerative diseases, while their immunomodulatory properties merit comprehensive investigation. Copper ions serve as cofactors for various enzymes and are involved in the regulation of immune cell activity and oxidative stress responses, thereby potentially affecting the functions of critical immune cells, such as dendritic cells (DCs). A copper-manganese (Cu-Mn) nanocomposite demonstrates therapeutic potential by selectively releasing Cu2+ in the acidic tumor microenvironment (TME). The localized accumulation of Cu2+ induces cuproptosis in cancer cells, promotes the release of damage-associated molecular patterns, enhances DC activation - evidenced by a 2.3-fold increase in CD80/CD86 expression - and facilitates increased infiltration of CD8+ T cells (1.8-fold) in murine melanoma models, thereby augmenting anti-tumor immune responses. Utilizing the enhanced permeability and retention effect, the nanocomposite achieves intratumoral Cu2+ concentrations 5.2-fold higher than those attained with free chelators, without inducing off-target toxicity, as indicated by normal liver enzyme levels (210).
Importantly, DCs also play a critical role in maintaining immune tolerance and their dysfunction has been associated with the development of autoimmune disorders. In recent years, exogenously derived tolerogenic DCs (tolDCs) have emerged as a novel cell-based immunotherapy approach, designed to restore immune tolerance in patients with autoimmune disorders. For instance, Jonny et al (47) conducted a systematic review on the therapeutic potential of tolDCs in autoimmune diseases, including systemic lupus erythematosus, demonstrating that the induction of tolDCs in vitro can effectively modulate T-cell responses and promote antigen-specific immune tolerance. This approach aligns with the objectives of copper-regulating therapies (47).
Recent reviews have highlighted the importance of metabolic complications in cancer (211-213), such as hypercalcemia, which affect a significant proportion of patients with advanced malignancies and contribute to morbidity and treatment challenges. While hypercalcemia involves dysregulation of calcium homeostasis often mediated by parathyroid hormone-related protein and osteoclast activation, it shares with copper dysregulation a common theme of metal ion imbalance in cancer pathophysiology. Understanding these parallel mechanisms may provide synergistic insights into targeting ion homeostasis as a therapeutic strategy. For instance, just as bisphosphonates and denosumab are used to manage cancer-related hypercalcemia by inhibiting bone resorption (143), copper-chelation and ionophore-based therapies are being explored to modulate copper levels and induce selective cancer cell death (214,215). This complementary approach underscores the broader potential of metal-targeting therapies in oncology.
Among metal elements that perform essential biological functions, such as iron, zinc, and copper, copper demonstrates distinct advantages due to its unique chemical properties. Copper, iron and zinc fulfil distinct roles in cellular redox processes. The Cu+/Cu2+ redox couple functions at a high reduction potential, facilitating efficient catalytic activity in essential enzymatic reactions such as cytochrome c oxidase-mediated mitochondrial respiration and SOD-dependent antioxidant defence. Unlike iron - which can induce non-specific oxidative damage through Fenton reactions - or zinc, which contributes to structural and catalytic stability without participating in redox cycling, copper supports tightly regulated electron transfer and dynamic, chaperone-mediated signaling processes via proteins such as Atox1. These unique properties position copper as a specialized redox messenger capable of mediating compartmentalized signaling pathways that are inaccessible to iron or zinc (216,217).

Research status of copper adjustment therapy
Copper-based targeted therapeutic strategies, including chelators, ionophores, and copper nanoparticles (CuNPs) (218,219), have made notable progress in clinical translational research (Table IV). Chelators primarily correct copper metabolic imbalances by sequestering excess copper ions (189), ionophores exploit copper-dependent pathological processes by facilitating the transport of copper ions across biological membranes (220) and CuNPs integrate the biological activity of controlled copper ion release with the targeted delivery capabilities of nanocarriers, thereby expanding their application scenarios in precision therapy (221).

Copper chelators
TM is a primary copper chelator under investigation for its role in copper-induced cell death. Preclinical studies indicate that TM exerts antitumor effects by inhibiting angiogenesis and directly suppressing tumor cell proliferation (222-224). Mechanistically, copper depletion - achieved either through TM treatment or knockdown of the copper transporter SLC31A1 - inhibits the progression of oral squamous cell carcinoma (OSCC) by selectively targeting cancer stem-like cells and reducing the stability of the histone methyltransferase EZH2. This identifies a novel 'copper-dependent proliferation' pathway that is essential for tumor growth. Notably, a recent clinical study evaluating TM combined with anti-PD-1 therapy in patients with locally advanced OSCC reported an objective response rate (ORR) of 57.8% and a 12-month overall survival (OS) rate of 80.0%, highlighting its potential to overcome immunotherapy resistance (225).
Beyond cuproptosis, copper also modulates ferroptosis. In pancreatic cancer models, copper promotes ferroptosis by inducing Tax1 binding protein 1-mediated autophagic degradation of GSH peroxidase 4 (GPX4). By contrast, copper chelators such as TM protect against ferroptosis-related tissue damage (e.g., in acute pancreatitis) by promoting GPX4 ubiquitination and aggregation through direct binding to cysteine residues (C107/C148) (226). Consistent with these findings, the chelator bathocuproine sulfonic acid enhances ferroptosis via copper depletion-mediated mitochondrial dysfunction and impairment of antioxidant defense (227).
Given copper's involvement in fibrosis and angiogenesis, TM has also been evaluated in related pathological conditions. In a bleomycin-induced pulmonary fibrosis model, TM downregulated VEGF expression and improved lung function (228). Its anti-inflammatory effects, mediated by the inhibition of copper-dependent cytokines, may further contribute to its antitumor efficacy by reducing the infiltration of pro-angiogenic immune cells (229). Additionally, NRF2 activation suppresses angiogenesis by inducing autophagy in vascular endothelial cells, thereby disrupting the energy supply to cancer cells (230).
The therapeutic potential of copper chelation extends beyond oncology. In patients with WD with reproductive dysfunction, engineered extracellular vesicles delivering a copper-chelating ligand significantly reduced non-ceruloplasmin-bound copper (42.7%) and urinary copper (47.7%), normalized gonadotropin levels in 87.5% of patients and exhibited a favorable safety profile (6.25% mild gastrointestinal events) (231). In oncology clinical settings, trientine combined with radiotherapy improved the ORR in hepatocellular carcinoma (65.5 vs. 37.9% with radiotherapy alone) and achieved a 12-month OS rate of 75.9% (232-234). For HER2-negative metastatic breast cancer (235-237), a polymer-based nano-copper chelator delivering paclitaxel achieved an ORR of 44.4% and a 6.8-fold increase in intratumoral drug accumulation (238). In case of drug-resistant Pseudomonas aeruginosa pneumonia, nebulized copper-chelator nanoparticles achieved an 82.1% sputum bacterial clearance rate and significantly shortened antipyretic time, while maintaining a favorable safety profile (239).

Copper ionophores
Elesclomol, a highly selective copper ionophore, binds Cu(II) to form a complex that is rapidly transported into mitochondria (184). Within the mitochondria, FDX1 facilitates the reduction and dissociation of the elesclomol-Cu(II) complex, leading to the generation of ROS (240). Notably, elesclomol also operates through an FDX1-independent pathway that enables an extracellular Cu(II) release pathway, distinguishing it from other ionophores (241). This sustained copper delivery mechanism results in a chronic elevation of intracellular copper and ROS levels, ultimately inducing cell death (242).
The anticancer effects of elesclomol have been demonstrated across various malignancies. In colorectal cancer (CRC) models, elesclomol promotes ATP7A degradation (243), leading to mitochondrial copper accumulation, oxidative stress and the subsequent degradation of SLC7A11 - a key component of the cystine/glutamate transporter - thereby inducing ferroptosis (178,184). Beyond CRC, a novel natural copper ionophore, Pochonin D (PoD), was identified in triple-negative breast cancer (TNBC). PoD covalently binds to Cys173 on peroxiredoxin 1 (PRDX1), inhibiting its peroxidase activity, promoting copper accumulation and ROS generation, and ultimately triggering copper-dependent cell death. This study unveils PRDX1 as a critical mediator in copper-induced cell death and highlights a new regulatory axis between copper metabolism and oxidative stress (244).

CuNPs
CuNPs have emerged as promising agents for therapeutic applications due to their tunable physicochemical properties and multifunctional capabilities. For instance, a CuO nanoplatform co-loaded with elesclomol potently induces copper-dependent cell death and enhances antitumor immunity. When combined with PD-1 inhibitors, this strategy achieves an 85% tumor suppression rate in melanoma models (219). In prostate cancer, the combination of elesclomol and CuCl2 induces DLAT aggregation and inhibits protective autophagy through mTOR activation, thereby synergizing with docetaxel chemotherapy (245). The application of CuNPs extends beyond oncology. Electrospun nanofiber membranes embedded with CuNPs exhibit broad-spectrum antibacterial activity against pathogenic Escherichia coli (246). Furthermore, CuNPs have demonstrated neuroprotective effects in models of AD by improving cognitive function (247), and ultra-small CuNPs embedded in hydrogels have been shown to significantly accelerate diabetic wound healing (248). Despite these promising findings, the clinical translation of CuNPs requires further optimization to address challenges related to long-term stability (249), targeting specificity and scalable manufacturing (250,251).

Challenges of copper adjustment therapy
In summary, copper-targeting approaches introduce novel concepts and hold substantial promise in disease diagnosis and treatment, representing a potential therapeutic strategy. However, further comprehensive research and validation are required for its clinical application to refine therapeutic protocols, enhance treatment efficacy and minimize adverse effects.

Dependence on transporters for drug uptake
The cellular uptake of copper-related therapeutics is tightly regulated by key transporters: CTR1 and the P-type ATPases ATP7A and ATP7B. As the primary mediator of copper influx, CTR1 also facilitates the entry of platinum-based drugs into tumor cells, a process that is critical for achieving therapeutic concentrations. By contrast, ATP7A and ATP7B act as copper efflux transporters, regulating the extrusion and subcellular sequestration of excess copper and platinum drugs (250). Dysregulation of CTR1 and ATP7A/B, whether through altered expression (e.g., CTR1 downregulation in drug-resistant tumors) or genetic mutations, disrupts copper homeostasis and impairs drug accumulation. For instance, ATP7A loss-of-function mutations cause MD (48), whereas ATP7B mutations underlie WD (251). In neurodegenerative diseases (such as AD), abnormal activity of copper-related transporters can disrupt brain copper homeostasis. Excess copper promotes Aβ protein aggregation and ROS overproduction, thereby exacerbating neuronal damage (79,252).

TME limitations
Copper-targeted therapy is significantly limited by the TME. In the hypoxic microenvironment of solid tumors (253), HIF-1α biaxially induces resistance to copper-dependent cell death by upregulating pyruvate dehydrogenase kinase 1/3-mediated degradation of DLAT and promoting MT2A-mediated chelation of free copper. This results in resistance to copper ionophores in nearly 60% of clinical samples, with the IC50 of elesclomol increasing by 7.2-fold under hypoxic conditions (254). Meanwhile, vascular abnormalities, elevated interstitial pressure and infiltration of immunosuppressive cells within the TME significantly impair drug delivery. For instance, in hepatocellular carcinoma, multidrug resistance mechanisms, such as the overexpression of ATP-binding cassette transporters, act synergistically with these TME-associated barriers to reduce intratumoral drug accumulation (255).

Long-term toxicity risks of chronic copper exposure
Copper, an essential micronutrient, maintains precise systemic homeostasis through coordinated regulatory mechanisms. However, long-term administration of copper-related therapeutics (e.g., copper ionophores and chelators), disrupts this balance. Excess copper catalyzes the generation of ROS via the Fenton reaction, causing oxidative damage to lipids, proteins and DNA, and ultimately leads to cytotoxicity (249).
Although cuproptosis induced by copper ionophores exhibits anti-tumor potential, preclinical studies have demonstrated that prolonged high-dose treatment elicits severe toxicity in normal tissues. For instance, mitochondrial-targeted copper-depleting nanoparticles effectively inhibit the growth of TNBC but still pose toxicity risks to healthy mice, thereby narrowing the therapeutic window (256). Additionally, the hypoxic TME promotes resistance to cuproptosis through HIF-1α activation, which further compromises therapeutic efficacy (254).

7.
Conclusions
Copper is the third most abundant trace element in the body after iron and zinc. It is essential for processes such as oxidative phosphorylation, free radical scavenging, angiogenesis, bone metabolism, neurotransmission and the synthesis of copper-dependent enzymes. On the other hand, both copper overload and deficiency can lead to impaired cellular function or even cell death, and their prolonged presence can trigger associated dysfunctions or neurodegenerative disorders such as hepatomegaly, MD, AD and PD.
It has been found that there are interactions among copper, iron and zinc, and the use of combination drug formulations for the treatment of related diseases may represent a new therapeutic strategy. In the treatment of diseases associated with copper overload, strategies such as reducing dietary copper intake, administering oral zinc to inhibit copper absorption or employing copper chelators to decrease intracellular copper levels may represent promising therapeutic approaches.
In the context of cancer, copper levels in cancer cells can exceed the tolerance threshold of normal cells, leading to significant adverse effects. Conversely, reducing intracellular copper levels can diminish the synthesis of the copper-dependent enzyme SOD1, thereby substantially impairing the organism's antioxidant capacity. Copper-induced cell death exhibits greater sensitivity in cells reliant on oxidative phosphorylation than in those dependent on glycolysis for energy production. Thus, converting the metabolic pathway of cancer cells from glycolysis to oxidative phosphorylation, coupled with exploiting the characteristic of elevated intracellular copper, may offer a promising new approach to cancer therapy. Comprehensive studies of the mechanisms of copper-induced cell death may help to identify more effective therapeutic agents, thus providing new insights and approaches for the treatment of related diseases and improving the prognosis of affected patients.
Copper regulation therapy has demonstrated significant potential in the diagnosis and treatment of diseases. Copper chelating agents, copper ion carriers and copper-based nanoparticles have shown therapeutic effects across various disease models. For instance, clinical studies have shown that the combination of the copper chelating agent TM with anti-PD-1 therapy achieves promising outcomes in the treatment of locally advanced OSCC. The copper ion carrier elesclomol exhibits anticancer effects in multiple malignant tumor models. CuNPs have yielded remarkable results in cancer therapy, antibacterial applications, neuroprotection and wound healing. However, the clinical application of this therapeutic approach faces several challenges. These include drug uptake dependence on key transporters such as CTR1, ATP7A and ATP7B, whose dysregulation can compromise therapeutic efficacy; the influence of the TME, including hypoxia and vascular abnormalities, which may limit treatment outcomes; and the potential toxicity associated with long-term copper exposure, which also narrows the therapeutic window.