Emerging Roles of Metallothioneins in Human Pathophysiology: A Review

ABSTRACT

Background

Metallothioneins (MTs) are small molecular‐weight proteins that either bind or release essential transition metals, depending on a specific cell situation. There are 4 major isoforms of MTs (1–4), of which MT1/2 is found in every cell in the body.

Aim

To provide a comprehensive discussion on the role of metallothionein in human pathophysiology.

Methods

This review was conducted using a variety of search engines, including Google Scholar, PubMed Central, Scopus, Web of Science, and others.

Results

The antioxidant property of MT is enhanced based on the availability of zinc. MTs reduce Pro‐inflammatory cytokine production and shift the Th17/Treg balance, ultimately alleviating inflammatory diseases. In neurodegenerative diseases, MTs interact with protein aggregates and modulate their formation and toxicity. The MT's molecular processes in neurodegenerative illnesses include metal ion control, antioxidant activity, and anti‐inflammatory actions. In cancer, MT acts as a tumor suppressor in the early stages and promotes tumor progression in the later stages. Only a few MT isoform‐targeting approaches have been approved for use as therapeutic drugs.

Conclusion

Metallothioneins play vital roles in controlling oxidative stress, immunity, metal homeostasis and protein aggregate for many diseases. Their dual roles as defenders in inflammation and neurodegeneration, and as suppressors and facilitators in cancer, highlight the complex nature of their biological role.

Abbreviations

AD

alzheimer's disease

AKt

protein kinase B

APT

adenosine triphosphate

AREs

antioxidant response elements

CNS

central nervous system

DSS

dextran sulfate sodium

GREs

glucocorticoid response elements

HCC

hepatocellular cancer

IBDs

inflammatory bowel diseases

IL

interleukins

KO

knockout

MFT1

metal response element‐binding transcription factor 1

MMP

matrix metalloproteinase

MREs

metal response elements

MS

multiple sclerosis

MTs

metallothioneins

NF‐κB

nuclear factor‐kappa B

NSCLC

non‐small cell lung cancer

PD

parkinson's disease

RA

rheumatoid arthritis

ROS

reactive oxygen species

SCC

squamous cell carcinoma

STAT

signal transducer and activator of transcription

TFs

transcription factors

TNF‐a

tumor necrosis factor‐a

VEGF

vascular endothelial growth factor

1. Background

Metallothioneins (MTs) are tiny proteins with a molecular weight of 6–7 kDa that either bind or release biological transition metals, depending on a specific cell situation. In 1957, Vallee and Margoshe discovered MTs as proteins that bind to cadmium in the renal cortex of horses [1]. Initial research concentrated on protein structure, but it quickly expanded to other functional and biochemical characteristics [2]. The human metallothionein (MT) gene cluster on chromosome 16q13 consists of several functional genes (MT1A‐MT1X, MT2A, MT3, MT4) and a number of pseudogenes, corresponding to a well‐conserved evolutionary structure which may correlate with coordinated gene regulation. Variation in the regulatory regions as well as in the coding region of the MT genes affects expression levels, the binding of metals, and is implicated in diseases like cancer and metal toxicity. Single nucleotide polymorphisms (SNPs) in the MT2A gene, notably rs28366003, rs10636, and rs1610216, are linked to disrupted metal homeostasis and increased risk of cancer, cardiovascular diseases, and type 2 diabetes, serving as potential biomarkers of metal toxicity and disease susceptibility [3, 4, 5]. MT plays a crucial role in transporting metals like copper and cadmium, regulating their metabolism, and protecting cells from the toxicity of unbound cadmium [3].

Metallothioneins can bind seven divalent metal ions, and this binding stabilizes their three‐dimensional structure. The MT is shaped like a finger, and it connects with the body when the metal level rises by activating these fingers. If the body has low levels of metal, the opposite occurs [1, 2, 3, 4]. MTs have long been known to regulate metal levels, preventing stress and heavy metal toxicity; however, emerging evidence shows their immunomodulatory effects influencing signal transduction pathways [6, 7]. MTs play key roles in metal detoxification, oxidative damage defense, DNA repair, cell proliferation, differentiation, neuroprotection, and apoptosis, with relevance to inflammatory diseases, neurological disorders, carcinogenesis, and chemotherapeutic development [6, 7, 8, 9].

Metallothionein provides zinc to activate transcription factors, crucial for the homeostasis of zinc‐dependent proteins which is essential for cell function and metabolism [10]. Zinc fingers, composed of cysteine, bind DNA bases and play roles in DNA recognition, transcription, RNA packing, protein folding, and apoptosis, regulating tissue development and neoplastic transformation [11, 12]. Because zinc availability is critical for a wide range of cellular activities, it is expected that MT dysregulation is linked to both normal and pathological conditions [13]. Studies show MT isoforms play a key immunoregulatory role in cancer [14], neurological disorders [11], and inflammation [10], making them a crucial immunotherapy target [10, 14]. Recent studies have explored the pathophysiological functions of metallothionein, but no comprehensive review has examined its significant role in various illnesses. As a powerful antioxidant and anti‐inflammatory, metallothionein can play an important role in a wide range of disease processes. A thorough understanding of metallothionein's function in various disease conditions is required for its full potential use. Therefore, this review aims to comprehensively discuss MT's role in human pathophysiology.

1.1. Cellular Localization and Biochemical Characteristics of Metallothioneins

Metallothionein is primarily synthesized in the liver and kidney and intestine and is found in high concentrations in fast‐dividing and transforming cells. Studies suggest that MT plays a key role in cell differentiation and proliferation [15, 16]. MT1 and MT2 are primarily cytoplasmic in non‐pathological tissues but are also detected in the nucleus, lysosomes, and the intermembrane space of liver mitochondria [15]. Their location varies during the cell cycle, with higher cytoplasmic expression in G0/G1, nuclear presence in S/G2, and cytoplasmic expression post‐G2/M transition [17, 18]. MTs translocate to the nucleus during liver regeneration post‐hepatectomy, likely reflecting their role in supplying zinc to transcription factors and enzymes for cell division [10, 19, 20].

MT translocation to the nucleus during the G1 to S phase transition likely protects DNA from free radicals and supplies zinc to enzymes involved in DNA replication, transcription, and RNA processing [21, 22]. MTs can diffuse into the nucleus via the nuclear pore complex due to their small size, with translocation requiring the small GTPase and other cytosolic molecules besides importins [23]. Additionally, reports exist regarding the import of MTs through the nuclear perinuclear cytoskeleton‐associated MT1 and MT2 mRNA. Effective protein shuttling at the G1 to S phase transition relies on MT1 and MT2 mRNA being anchored to the perinuclear cytoskeleton via eEF1 binding at nucleotides 21–36 and 66–76, enabling localized protein production [23, 24, 25].

Mammalian metallothioneins (MTs) are small proteins composed of 61 to 68 amino acids, with a high cysteine content and minimal histidine or aromatic amino acids [2, 4, 26]. MT isoforms are categorized based on factors like molecular weight, metal binding, and amino acid sequence [27]. MT1 and MT2 are expressed in all body cells, while MT3, primarily found in the CNS, plays a role in brain development and apoptosis and is found in normal skin and malignant skin lesions as well as breast and urinary bladder cancers [16, 28, 29]. MT4, located in the stratified epithelium, is involved in pH regulation and protection against skin injuries as well as contributing to taste and texture characterization on the tongue [16, 28, 29].

1.2. Metallothionein and Zinc

Metallothioneins play a crucial role in zinc homeostasis through the oxidation and reduction of cysteine sulfur ligands, which facilitate zinc release. Zinc and cadmium binding is essential to maintain the stability of MT; this ensures intracellular zinc availability [30, 31]. Zinc‐bound MT sources zinc from cellular pools, influenced by diet and transporter function, and can donate or extract zinc for metalloproteins [32]. Apo‐MT prevents DNA binding of zinc‐finger transcription factors like Spl and TFIIIA, while Zn‐MT restores activity [9, 13]. MTs regulate apoptosis, cell division, and gene expression by supplying zinc to proteins and transcription factors. During oxidative stress, they scavenge free radicals, enhancing zinc release and forming MT‐disulfide. This disulfide form is unstable and converts back to the thiol form in a more reduced environment, aided by selenium and elevated GSH/GSSG levels [32, 33]. Consequently, MT is essential to avert glutathione depletion. MT's superior antioxidant capacity over GSH is not just due to its higher thiol content, but also to its unique cysteine‐cluster structure, which enables efficient free radical scavenging and redox cycling [33].

1.3. Metallothionein Expression and Regulation

Despite not having a classical signal peptide MT can be released under stress through nonclassical pathways adding to the extracellular pool found in tissue spaces milk urine and serum. Using receptors like megalin (LRP‐2) and CD91 (LRP‐1) this extracellular MT can alter inflammatory and immune responses. Furthermore MTs ability to both respond to and induce the expression of Pro‐inflammatory cytokines (IL‐1 IL‐6 and TNF‐α) emphasizes a reciprocal regulatory role in cytokine signaling and inflammatory pathophysiology [34]. In response to tissue injury, infection, inflammation, and neoplastic illness, MT expression can be altered by physiological mediators through response elements in the promoter [35]. Cytosolic MT levels can increase up to 100 times after inflammation, sequestering zinc. MT induction occurs within 2–4 h and depends on zinc availability [35, 36, 37]. MT gene expression is regulated by elements like the TATA box, GC box, glucocorticoid response elements (GREs), antioxidant response elements (ARE), and metal response elements (MREs), which interact with TFIID, AP1, and AP2 and are crucial for metal induction [8, 32, 37, 38]. Zinc activates MTF1 by displacing its inhibitor, allowing it to bind to MREs in the MT promoter, promoting MT transcription. MTF1 translocates to the nucleus after zinc activation, and its activity is regulated by phosphorylation/dephosphorylation, with protein phosphatase 2 A (PP2A) controlling MT production [39, 40, 41, 42, 43]. Zn is the only metal that activates metal response element‐binding transcription factor 1 (MTF1) [44]. Other heavy metals, like cadmium and lead, can promote MT transcription via MRE. MT gene expression is regulated by the TATA box, response elements like ARE and MRE, and factors like Sp1, TPA, USF, Ap1, Ap2, and MTF‐1 (Figure 1) [7, 35, 37].

Figure 1.

A summary of the regulation and function of the metallothionein (MT) gene [35]. Many response elements in the MT promoter up‐regulate transcription. These are some examples: 1) following zinc occupancy, MTF‐1 activates MRE at the promoter region, which is determined by zinc from dietary supply; 2) glucocorticoid response elements (GRE); 3) elements that are triggered by cytokine signaling through STAT proteins; and 4) antioxidant response elements (ARE), also known as electrophile response elements, which are triggered by redox conditions. Methylation also suppresses expression in certain tumor cells; 5) Release of Zn from Zn7MT to zinc‐dependent apoproteins, indicating MT's role in metal homeostasis.

2. The Role of Metallothioneins in Diabetes

Metallothioneins, notably MT1 and MT2A, exert opposing and context‐dependent effects on pancreatic beta‐cell function and diabetes. In the transgenic mice overexpressing the human MT2A gene under the insulin promoter (MT2A‐Tg) models, MTs have been shown to possess a protective antioxidant activity during STZ‐induced hyperglycemia and islet viability, which protects against oxidative and nitrosative damage [45, 46, 47]. These protective effects lead to better islet graft survival and glycemic control in transplantation animals. Similarly, exogenous administration of recombinant MTs fused to cell‐penetrating peptides, such as Tat‐MT1A, provides resistance to glucolipotoxicity, hypoxia, and cytokine‐induced oxidative stress in beta cells in vitro and in vivo [48]. However, contradicting evidence from nonobese diabetic (NOD) mice suggests that MT2A overexpression can speed up diabetes onset, particularly in males, despite decreasing cytokine‐induced ROS. This harmful effect is connected to more beta‐cell death, weaker insulin signaling (because of less AKT/FOXO1 phosphorylation), and lower levels of PDX1, suggesting that MTs might change key survival and function processes without relying on their antioxidant properties [49, 50].

2.1. The Role of Metallothioneins in Oxidative Stress and Inflammatory Diseases

Metallothioneins (MTs) neutralize free radicals through redox‐dependent mechanisms, with thiolate ligands in cysteine residues contributing to antioxidant activity via oxidative stress‐induced zinc mobilization [2, 4]. Rising oxidative stress promotes MT gene expression, which allows for ROS scavenging [8, 34, 35, 36], lysosomal stabilization, and decreased apoptosis [47]. MTs surpass glutathione at lowering ROS and preventing lipid peroxidation [51, 52]. Inflammation, infection, and stress all activate MTs, which behave similarly to acute‐phase proteins. Lipopolysaccharide (LPS) enhances MT1, particularly in the liver, whereas MT‐null animals exhibit immunological dysfunction [2, 9, 10]. MTs control immunological responses by modulating zinc‐dependent proteins such as p53, NF‐kB, and Sp1 [37, 38]. LPS stimulates MT1 production via IL6RE and glucocorticoid synergy near the MT1 promoter [53, 54], and IL‐6‐induced STAT phosphorylation mediates MT's anti‐inflammatory actions [55, 56]. MT‐KO mice show greater tissue injury and cytokine levels [54, 57, 58, 59]. MTs reduce inflammation by disrupting NO formation and facilitating zinc withdrawal from microbes, supporting antibacterial function [22, 60]. Extracellular MTs act as chemokine‐like danger signals despite lacking classic structure due to high cysteine content [61, 62].

2.2. The Role of Metallothionein in Rheumatoid Arthritis Pathogenesis

Rheumatoid arthritis (RA) is an inflammatory disease that commonly affects tissues and joints [63]. MT has functional effects on immune cells and may play a role in preventing RA as a stress response protein that sequesters toxins. Studies have revealed that MT1 expression is highly elevated in RA and is directly linked to disease progression. Similarly, when MT1 was administered locally, it significantly decreased pathogenic signs and synovial inflammation in rheumatoid mice [64, 65]. An imbalance between Th17 and Treg cells leads to RA development, with Th17 causing joint destruction and Tregs preserving immunological tolerance [66, 67]. MT1 reduces RA inflammation by lowering Pro‐inflammatory cytokines and shifting CD4 + T cell development to Treg cells while decreasing Th17 cells [66]. This shift occurs through STAT3 pathway suppression, which suppresses STAT3‐mediated RORγt activation and Th17‐related cytokines [54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66]. MT1 also controls Th1 responses in autoimmune and arthritic diseases [54].

Evidence from MT1 and MT2 deficient and transgenic mice suggests that in autoimmune arthritis, MT1 may be crucial for controlling the Th1 response [68]. In addition, in mice with developed arthritis, daily injections of MT1 and MT2 proteins significantly reduced disease occurrence and severity [66]. Furthermore, MT directly inhibits the expression of RA‐related inflammatory cytokines throughout the pathogenesis of RA. Pro‐inflammatory cytokines, such as IL‐6, IL‐1, IFN‐γ, IL‐17, and TNF‐α, which are key mediators in RA progression, were significantly suppressed in the peripheral blood mononuclear cells of RA patients after treatment with recombinant MT1, while this phenomenon was not observed in the control group [65, 69]. Evidence from these studies suggests that MT1 may have potential therapeutic value for this disease [54].

2.3. Metallothionein and Inflammatory Bowel Diseases

Inflammatory bowel disorders (IBDs) have a complex pathophysiology involving immune system disorders, environmental factors, gut microbiome changes, and local immune abnormalities [70, 71]. IBD patients exhibit increased pro‐inflammatory cytokine release, oxidative stress, and altered apoptosis [56]. MTs contribute to IBD inflammation, with animal studies showing significantly higher MT1 and MT2 expression in inflamed colitis tissue. MT1 and MT2 deficiency worsens DSS‐induced colitis, while MTs help prevent mucosal inflammation by reducing pro‐inflammatory cytokine production [56, 72]. In MT1/2 (‐/‐) KO mice, colitis induction caused greater myeloperoxidase and cytokine production than in wild‐type mice, indicating MTs' protective role in reducing inflammation [68].

IBD patients have weakened antioxidant barriers and severe local ROS activity, correlating with disease activity [73, 74]. MT upregulation may defend against ROS damage [56, 75], yet local MT expression in IBD epithelium is lower than in healthy individuals [76]. However, some studies found MTs non‐protective, with MT‐null mice showing reduced disease markers post‐DSS treatment [77, 78, 79]. Further studies reported no significant differences in mucosal MT expression among IBD patients [80, 81] nor genetic variations predisposing to IBD [82]. MTs reduce disease severity by inducing anti‐inflammatory cytokines like TGF‐β and IL‐10. In all animal models, MTs act as barriers to local inflammation [83, 84]. MTs help reduce IBD severity by reducing inflammation, modulating oxidative stress, immune responses, apoptosis, and zinc homeostasis through antioxidant, anti‐inflammatory, and Antiapoptotic functions [56, 77, 78, 79, 83].

2.4. Metallothionein in Multiple Sclerosis Pathogenesis

Multiple sclerosis (MS) is an inflammatory demyelinating condition of the central nervous system (CNS) with complex pathophysiology [85]. Oxidative stress promotes demyelination and neuronal degeneration in MS, and MT plays a key role as an antioxidant. MT1 and MT2 expression is elevated in MS brain lesions, particularly in macrophages, microglia, and reactive astrocytes, with higher expression in inactive lesions, suggesting a role in alleviating the disease [86, 87]. MT maintains zinc homeostasis, and zinc deficiency disrupts immune cell function, leading to lower metallothionein levels, reduced innate defense, and T‐cell‐mediated autoimmunity [9, 10, 54, 88]. Zinc deficiency also affects Th17 downregulation and the balance of T cells in MS [66, 68].

MT1/2 levels are elevated in MS and experimental autoimmune encephalomyelitis (EAE) [89, 90]. MT1 and MT2 knockout mice are more susceptible to disease [91], and inflammatory cytokines likely control MT production in MS lesions [92, 93, 94]. In EAE, MT1 and MT2 deficiency increases T cell and macrophage infiltration in the CNS and promotes pro‐inflammatory cytokines like TNF‐a, IL‐6, and IL‐1b. MT therapy can reduce EAE progression, alleviate tissue loss, and reduce CNS inflammation. MT2 treatment reduces symptoms and inflammation in EAE mice, while MT1 has antiapoptotic and antioxidant effects [93, 94, 95].

MT2 primarily promotes disease remission in EAE, while MT1 has minimal impact. MT1 influences serum cytokine patterns, and MT2 boosts Th2 responses and preserves myelin in the spinal cord, with less WBC infiltration in MT2‐treated mice [92]. MT2's stronger Zn‐thionein character maintains Zn (II), while MT1's lower Zn (II) properties allow for Cu (I) binding [91, 96].

2.5. Metallothioneins in Neurodegenerative Diseases: Neuroprotection and Metal Homeostasis

A high oxygen consumption by the central nervous system (CNS) makes it highly vulnerable to oxidative stress [97]. By scavenging reactive oxygen species and binding toxic metal ions, MTs are essential for cellular defense against oxidative stress and heavy metal toxicity [97, 98, 99]. Their potential as both neuroprotective agents and indicators of metal‐induced neurotoxicity is highlighted by their overexpression in the central nervous system, especially when exposed to heavy metals like Pb, Cd, and Hg over an extended period of time. The antioxidant effects of MT play an important role in tackling the clumping of altered proteins and their toxicity [96, 97, 98, 99].

Reactive oxygen species play a central role in AD due to limited brain regeneration. MT1/2 are upregulated near amyloid plaques, especially in the hippocampus and cortex, suggesting involvement in AD pathogenesis [97, 100, 101, 102, 103]. MT1 suppresses Aβ‐induced microglial activation and neurotoxicity [104], while disrupted zinc homeostasis and elevated Zn and Fe levels in affected brain regions contribute to pathology [7, 105]. MT2A inhibits Aβ aggregation and reduces inflammation and neurotoxicity [7, 101, 102, 103]. MT3, though protective, is downregulated in AD; it blocks Aβ1‐40 toxicity and prevents harmful aggregates [7, 97, 106, 107, 108]. MT3 also scavenges Aβ via transthyretin binding [97, 98], and Zn7MT3 neutralizes redox‐active copper, preventing ROS and neuronal damage [109, 110, 111, 112]. Both Zn7MT3 and Zn7MT2A prevent Cu‐Aβ1‐40‐induced neurotoxicity [113]. MTs inhibit apoptosis by blocking caspase‐3 and DNA fragmentation [6, 7, 99, 100, 101]. The protective effect of human Zn7MT3 against copper‐mediated toxicity in neurodegenerative diseases is summarized (Figure 2).

Figure 2.

Metal‐swap reactions and Cu(II) redox‐silencing by Zn7MT3 in neurodegenerative diseases [100]. The metal switch between Zn7MT3 and the disease‐specific amyloidogenic Cu(II)‐Aβ1‐40 peptide, Cu(II)–α‐Syn, and Cu(II)‐PrP proteins eliminates ROS generation and the cellular toxicity that goes with it. Cu(II) is reduced by the thiolates as it binds to the N‐terminal domain, creating the Cu(I)4Zn4MT‐3 species as well as the non‐redox‐active Zn(II)‐Aβ1‐40, Zn(II)‐‐Syn, and Zn(II)‐PrP. The N‐terminal β‐domain of Cu(I)4Zn4MT‐3 contains an air‐stable Cu(I)4‐thiolate cluster and two disulfide residues.

Metallothioneins also play a role in the etiology of Parkinson's disease (PD), providing neuroprotective benefits via metal chelation, antioxidative action, and regulation of microglial activation [111]. PD is characterized by the gradual loss of dopamine neurons that provide signals to the striatum [114]. In PD, dopaminergic neurons can produce excessive dopamine, leading to neurotoxicity. Intrinsic MT may protect against dopamine quinone‐induced neurotoxicity in hemiparkinsonian animals lacking MT1 and MT2 receptors [115]. In an animal model of PD, MT2 treatment exhibited a neuroprotective effect that mitigates rotenone‐induced neurodegeneration [116]. Levodopa increased MT3 mRNA expression in the undamaged striatum, suggesting a link between MT3 regulation and progressive degeneration in PD, highlighting the role of metallothioneins in dopaminergic dysfunction [117, 118, 119].

In laboratory experiments investigating the interaction of Zn7MT‐3 with copper (II)‐bound Syn and prion proteins, it was discovered that Zn7MT‐3 acts protectively by sequestering copper [120]. The protein's thiol groups simultaneously reduce Cu(II) while binding to the N‐terminal region, leading to the formation of the Cu(I)₄Zn₄MT‐3 complex and non‐redox‐active Zn(II)‐Aβ1‐40, Zn(II)‐α‐Syn, and Zn(II)‐PrP species [97]. Human MT1A, transduced into mitochondria, restored tyrosine hydroxylase expression, improved mitochondrial function, reduced ROS, and alleviated dopaminergic neuron degeneration in PD, suggesting a potential treatment option [121].

Studies have also shown that MT induction aids in the production of coenzyme Q10 in the brains of transgenic and KO mice in PD models. This is accomplished by activating lipoamide dehydrogenase, which improves coenzyme Q10's antioxidant capacity [122]. Ubiquinone reduces pro‐inflammatory cytokines and boosts the production of ubiquinol but decreases synuclein nitration [122]. Likewise, investigations in vitro as well as in vivo revealed that MT gene overexpression decreased the amount of ROS produced, caspase‐3 activation, and lipid peroxidation caused by lipid peroxidating agents [123, 124]. The therapeutic applications of MTs and MT‐derived peptides for reducing metal‐induced misfolded protein aggregation and inflammation make them a possible therapeutic target in neurodegenerative illnesses like PD [111, 120].

3. Roles of Metallothionein in Carcinogenesis

Metallothioneins may promote tumor growth through mitogenic actions and apoptosis suppression. MT overexpression has been associated with resistance to anticancer medicines and radiotherapy [19, 20, 32]. MTs are essential to tumor cell viability because they combat metal toxicity, oxidative stress, and proliferation [3, 21]. In some situations, MT expression is related to more aggressive and higher‐grade tumors, whereas in others, it's related to more differentiated, lower‐grade tumors [16]. Reduced MT expression has been seen in various cancer types, including liver [16, 25, 125], colon [126], and prostate [14]. Besides, the expression of MT decreases gradually throughout the change from normal colorectal tissue to adenomatous polyps and cancer [127]. In contrast, abnormal MT overexpression has been discovered in malignancies, including breast cancer and oral squamous cell carcinoma [13, 16, 32, 128, 129].

Overexpression of MT has a role in medication resistance because nuclear expression of MT protects DNA in cancer cells from the damaging effects of therapies [16, 17, 20]. Indeed, MT knockout mice were found to have a greater rate of induced carcinogenesis [16]. There is proof that MT1/2 both offer protection against chemical carcinogens. After 14 weeks of 7,12‐dimethylbenz[a]anthracene (DMBA) therapy, MT‐/‐ mice developed cutaneous tumors, but MT + /+ mice did not. These MTs were discovered to be endogenic protection factors against DMBA‐induced skin malignancies [16, 130]. In cancer, MT has a dual function, suppressing tumor growth in the early stages and promoting tumor progression in the later stages [131, 132, 133].

3.1. Metallothioneins and Tumour Progression

Tumors with elevated MT expression may have enhanced the breakdown of extracellular matrix components and facilitated tumor cell invasion [129]. The possible antiapoptotic action of MT due to its regulatory action on p53 or NF‐κB activity by donating or withdrawing zinc ions may be connected to a less favorable prognosis in individuals with increased MT expression [134, 135, 136, 137]. MTs remove zinc from P53, favoring an aggregation‐prone state that leads to p53 instability, inactivation, and loss of function. As a result, uncontrolled cell growth occurs. Overexpression of MTs had a strong inhibitory effect on P53 transcriptional activity, which results from the metal chelation function of MTs [138, 139]. MTs interact with NF‐κB and alter its antiapoptotic actions, most likely because zinc is required for NF‐κB's DNA‐binding function [135]. MT2A also influences the activation of the NF‐κB pathway and thereby contributes to tumor aggression in gastric and colorectal cancers [140, 141]. By increasing NF‐κB activity, MT1M downregulation can also aid in hepatocellular cancer development [142].

Persistent apo‐MT expression in tumor cells contributes to faster growth and survival of these cells by inducing inactive p53 [137]. Metal‐free MT interacts with p53 in tumor cells, preventing DNA binding and affecting transcription and apoptosis, suggesting an oncogenic role [143, 144]. MT1 and MT2 null cells have been documented in vitro and in vivo to be more susceptible to apoptotic stimuli and to have roughly 3–4‐fold greater basal levels of normally functioning p53 than counter‐MT‐containing cells [143]. Besides, following cisplatin treatment of cells from MT1 and MT2 gene KO mice, the amounts of p53 and the death effector protein Bax were much higher than in normal cells. Thus, MT deficiency promotes apoptosis susceptibility [145, 146].

Other researchers, however, have discovered no link between MT1 and 2 protein concentrations and p53 expression in cancerous tumors [147]. MT1 and 2 induction was greater in well‐functioning p53‐containing cells [148]. Apo‐MT removes Zn from p53, inactivating it, while Zn‐bound MT1 and 2 supports p53 function, highlighting the role of MT form, timing, and dosage in cancer. Elevated MT1 and MT2 levels reduce apoptosis in various cancers [147, 149]. MT1 and MT2 has been established to prevent apoptosis by increasing bcl‐2 and c‐myc, which are antiapoptotic oncogenes, while inhibiting proapoptotic tumor suppressor proteins such as caspase‐1 and‐3 and cytochrome c leakage [150]. The fact that Zn also impacts caspase‐3 activity may explain the link between MT1 and MT2 levels and caspase‐3 [23, 134].

MT1E promoter methylation is likely in breast cancer; a process of gene inactivation leads to decreased MT1E expression [150]. Moreover, cancer development has been correlated with MT1G suppression in esophageal, liver, thyroid, and prostate cancers. Promoter hypermethylation has been connected to MT1G silencing [151, 152]. The downregulation of MT1G in hepatocellular malignancy was likewise caused by an allelic loss on chromosomes 16q12.1–q23.1 [153, 154]. MT1G stabilizes p53 by inhibiting MDM2 and donating zinc, enhancing its activity. MT1G mutation induces Bax‐mediated cell death and p21‐driven arrest, potentially preventing HCC tumors [154, 155, 156, 157, 158].

CpG island hypermethylation promotes gastric cancer by inactivating MT3, MT1H, and MT1G [138]. The dephosphorylation of the C/EBP transcription factor in liver cancer suppresses MT1 and MT2A genes through the PI3K/AKT pathway. In thyroid cancer, MT1G has anticancer effects by reducing cell invasion and proliferation and promoting cell cycle termination and cell death. This is achieved by suppressing Akt and Rb phosphorylation or altering the PI3K/AKT and Rb/E2F signaling pathways [159, 160]. Zinc ions promoted MT isoform synthesis in breast cancer cells while also marginally increasing VEGF expression [161]. MT1/2‐deficient mice that produce IL‐6 demonstrated diminished IL‐6‐induced angiogenesis, showing that MT1/2 plays an angiogenic role through angiogenic molecule regulation [162]. MTs activate gelatinase (MMP), which is required for ECM breakdown and metastasis [163]. MMP‐9, which is connected to neovascularization and ECM remodeling, interacts with MTs and promotes tumor growth [164, 165].

3.2. Metallothionein 1 Modulated Signaling Pathways in Cancer

The inhibitory impact of MT1G on tumors in cancer depends on the PI3K/Akt pathway. Reduced Akt activation by MT1G enhances Mdm2 overexpression and suppresses cell adhesion, crucial in thyroid carcinogenesis [166]. Additionally, genes related to the Wnt/β‐catenin pathway were enriched in HCC patients with reduced MT1H expression. MT1H overexpression in HepG2 and Hep3B cells downregulates Wnt/β‐catenin target genes by lowering β‐catenin nuclear translocation, suppressing the pathway, and reducing tumorigenicity [167].

MT1 may have dual roles in NF‐kB activation. MT loss decreases the nuclear p65 subunit in fibroblast cell lines without affecting NF‐kB transcriptional function, increasing apoptosis susceptibility. Restoring MT1 expression elevates p65 levels, NF‐kB activity, and apoptosis resistance, making MT1 essential for NF‐kB activation [57, 168]. Similarly, MT overexpression enhances NF‐kB DNA binding and gene activation in breast tumor cell lines. However, MT1 gene transfection into MT‐KO cells inhibits TNF‐a‐induced NF‐kB‐dependent gene expression by preventing IkBa degradation [169]. MT1M expression also blocks TNF‐induced IkB degradation and NF‐kB transactivation in HCC, suggesting MT1 deficiency promotes carcinogenesis by increasing NF‐kB activity [140]. Zinc and zinc ionophores reduce NF‐kB DNA binding in HeLa cells, indicating MT1's role in NF‐kB signaling via zinc sequestration (Figure 3) [170].

Figure 3.

Signaling pathways that MT1 controls [57]. MT1 inhibits STAT1/3 phosphorylation, impeding the development of Tr1 cells that produce IL‐10. MT1 also suppresses Th17 cell proliferation by decreasing STAT3 phosphorylation. The elevation of p50/P65 activity by MT1 can positively affect the NF‐kB signaling pathway, which promotes the motility of tumor cells, tumor cell invesion, and cell death resistance. MT1 can also inhibit carcinogenesis by negatively regulating TNF‐a‐induced IkBa degradation. These fundamental activities are facilitated by MT1‐mediated suppression of free Zn2 + . By inhibiting Akt phosphorylation, MT1 inhibits the PI3K/Akt pathway. MT1 inhibits the Wnt/b‐catenin pathway by decreasing b‐catenin nuclear translocation.

3.3. Metallothionein Isoforms in Various Types of Cancer

MTs have a function in the onset of cancer and its course, but their expression is not ubiquitous in all human malignancies [14, 16, 25, 125, 126, 127, 128, 129]. So far, the underlying molecular processes that underlie the upregulation of MT expression in certain cancers and downregulation in others are not yet understood. Changes in isoform expression patterns could be one such explanation. Therefore, it was discovered that overexpression of particular MT isoforms affects the proliferation of low‐MT‐expressing cancer cells [5]. MT3 isoforms and all MT1 sub‐isoforms except MT1B are found in breast cancer. MT2A is upregulated in renal tumors, while MT1A and MT1G are downregulated. MT3 is present in renal carcinoma and normal kidney tubules but absent in normal bladder tissue, making it a potential bladder cancer biomarker. Bladder cancer also shows high MT1X mRNA expression [26, 171]. While the MT1X isoform was downregulated in cases of advanced prostate cancer, the MT1A, E, X, and MT2A isoforms were all prevalent in healthy prostate tissue. Additionally, MT1 and MT2 isoforms have been linked to the proliferative activity of human malignancies in the breast, colon, and prostate [26].

A study found that MT1X overexpression in HCC cells slows the cell cycle, enhances apoptosis, inhibits tumor development, promotes lung metastasis, and stops cell division through NF‐kB signaling [158]. In gastric adenocarcinoma, elevated levels of MT1X were observed in cancer‐associated fibroblasts, which subsequently enhanced angiogenesis [172]. MT1M is expressed in a range of typical hepatic tissues, with hepatic tissues having the greatest expression. MT1M expression, however, was significantly reduced in HCC specimens. According to a methylation profile investigation, most of the HCCs that have been investigated have methylated MT1M promoters [173]. MT expression in normal lung tissue appears to protect against potentially carcinogenic causes [174]. However, high‐MT lung epithelial cells are valuable for the subsequent activation of oncogenesis. Thus, it appears that MTs protect lung cells from harmful substances until a key event occurs, at which point they contribute to tumor formation [158, 175]. MTs' expression in tumors influences metastasis, cell adhesion, invasion, and migration. Understanding these changes can help identify treatment goals and inhibit tumor development [49, 176].

Although MT isoforms and sub‐isoforms (Table 1) have been proposed as diagnostic and prognostic biomarkers, there are limitations in their clinical utility because MT proteins can be induced by virtually any exogenous stressor (such as heavy metals, oxidative stress, inflammation, or exposure to drugs) thereby precluding disease specific interpretations. For example, increased MT levels could simply be a mark of cellular stress more generally, not a specific disease such as cancer. As suggested by previous studies, optimizing the isoform‐specificance detection method and combination with other onco‐markers is necessary to enhance detection sensitivity and specificity for diagnosis in clinical practice [3, 171].

Table 1.

Summary of Metallothionein Isoforms in Cancer.

MT isoform	Function/role	Regulation	Cancer‐related effects	References
MT1 (general)	Zinc homeostasis, Antiapoptotic, redox regulation	Induced by metals, stress, NF‐κB, methylation status	Promotes tumor survival, inhibits p53, modulates NF‐κB and PI3K/Akt signaling, enhances angiogenesis	[129, 134, 135, 136, 138, 139, 140, 141, 162, 164]
MT2A	Similar to MT1; regulates NF‐κB	Upregulated in renal tumors; PI3K/AKT involvement	Promotes invasion, survival; activates NF‐κB in gastric and colorectal cancers	[140, 141, 165]
MT3	Brain‐specific; growth‐inhibitory in normal tissue	Inactivated by promoter methylation in gastric cancer	Silencing contributes to cancer progression	[138, 171]
MT1E	Antiapoptotic, zinc donor	Promoter methylation in breast cancer → inactivation	Downregulation contributes to tumor progression	[150]
MT1G	Stabilizes p53, inhibits MDM2, enhances p21 and Bax	Silenced via hypermethylation and chromosomal loss	Tumor suppressor in liver, thyroid, prostate, and esophageal cancers	[151, 152, 153, 154, 155, 156, 157, 158]
MT1H	Regulates Wnt/β‐catenin, suppresses proliferation	Downregulated in HCC; promoter methylation	Tumor suppressor; its loss enhances β‐catenin activity	[159, 167]
MT1M	Regulates NF‐κB, suppresses tumor growth	Downregulated in HCC; promoter methylation	Inhibits TNF‐α‐induced NF‐κB activation, acts as tumor suppressor	[140, 142, 173]
MT1X	Modulates NF‐κB, apoptosis, and cell cycle	Overexpressed in HCC and gastric cancer fibroblasts	Suppresses tumor growth; enhances angiogenesis in fibroblasts	[158, 172]

3.4. Metallothioneins and Their Roles in Chemoresistance

Tumor MT expression is linked to both innate and acquired resistance to antitumor drugs. MTs contribute to chemoresistance by deactivating and chelating drugs, protecting biomolecules from cytotoxicity, and potentially driving multidrug resistance (MDR) [5, 32, 177]. Drug or metabolite sequestration by MT may restrict the antineoplastic medications' ability to interact directly with their intracellular targets, lowering the effectiveness of their actions. As a result, MT expression in malignant cells shields them while increasing tumor growth rate, resulting in shorter patient survival [5, 178]. Moreover, the antiapoptotic effects of MTs and their transcription factor (de)activation and ROS scavenging are favorable for the survival and growth of cancer cells, besides their protection against the immune system of the host [140, 141, 142, 143, 144]. Thus, MTs are double agents; they have significant implications for physiological mechanisms as well as cancer, providing resistance to antineoplastic drugs [5, 158, 179, 180, 181]. For instance, MT1G overexpression leads to sorafenib chemoresistance in HCC by suppressing non‐apoptotic regulated cell death, ferroptosis, which participates in glutathione depletion, lipid peroxidation, and iron metabolism [181]. MT‐3 promotes cisplatin resistance in neuroblastoma cells, revealing its role in tumor‐specific chemoresistance mechanisms [177]. It follows that the possibility of using tumor cell expression of MT to tailor treatment strategies is not surprising [158, 182].

3.5. Metallothioneins as Cancer Therapeutic Targets

MTs have beneficial effects for alleviating inflammatory and neurodegenerative diseases. However, MTs are double agents for cancer progression [5, 53, 158]. Because of their nucleophilicity, MTs have to shield cells from the lethal consequences of electrophilic antitumor medications. Depending on the isoforms of MTs, therapeutic options for targeting them include DNA methylation, siRNA, antisense mRNA, and microRNA. Therefore, MT gene KO is becoming more and more popular as a therapeutic objective for malignant tumors. One common method for permanently reducing gene expression is RNA interference [158, 183, 184]. To target MT2A in ovarian cancer, researchers created pRNA and siRNA chimeras. The pRNA/siRNA complex may limit MT2A expression, reducing cell proliferation. Thus, siRNA‐mediated silencing of the MT2A gene limits breast and ovarian tumor cell proliferation [185]. Conversely, MT1G overexpression in gastric cancer played tumor suppressive roles. It suppresses rapid proliferation, metastasis, and invasion by promoting feroptosis and autophagy, thereby inhibiting gastric cancer cell function and providing novel mechanisms and therapeutic alternatives [186]. To date, only a few MT isoform‐targeting approaches are approved for use as therapeutic drugs [5, 187]. More study is required to develop and refine these technologies, as well as to determine the clinical safety of different MT‐modulating approaches. Targeting specific MT isoforms in a range of malignancies suggests a bright future for MT biological applications in cancer treatment [5, 158].

4. Concluding Remarks

The antioxidant property of MT is enhanced based on zinc's accessibility. MTs reduce pro‐inflammatory cytokine production and shift the Th17/Treg balance, ultimately alleviating inflammatory diseases. In neurodegenerative diseases, MTs interact with protein aggregates and modulate their formation and toxicity. The MT's molecular mechanisms in neurodegenerative diseases involve its regulation of metal ion levels, antioxidant properties, and anti‐inflammatory effects. MT plays two roles in cancer, suppressing tumors in the early stages while promoting tumor progression in the later stages. Only a few MT isoform‐targeting approaches have been approved for use as therapeutic drugs. More study is required to develop and refine these technologies and assess the level of clinical safety of different MT‐modulating approaches.

5. Perspectives

In the future, the focus should be put on the generation of isoform specific metallothionein targeting compounds, based on the observation that metallothioneins have diverse functions in various pathophysiologies. Developments in high‐throughput screening and structural biology may speed discovery of safe and effective MT‐directed therapeutics. Studying MT functions in conditions other than those currently investigated may uncover new modes of action and therapeutic time windows, especially in neuroinflammatory and neurodegenerative diseases. Combining MT modulation with established therapies might improve disease course by focusing attention on the importance of a collaborative strategy in translating MT biology to the clinics.

Author Contributions

Ousman Mohammed: conceptualization, writing – original draft. Abdisa Tufa: resources. Solomon Tebeje Gizaw: investigation, writing – review and editing, writing – original draft, supervision, resources, project administration.

Ethics Statement

The authors have nothing to report.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Transparency Statement

The lead author Ousman Mohammed affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.

Mohammed O., Tufa A., and Gizaw S. T., “Emerging Roles of Metallothioneins in Human Pathophysiology: A review,” Health Science Reports 8 (2025). 10.1002/hsr2.71279.

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.