GGT, a core component of the γ-glutamyl cycle (21), has long been associated with GSH degradation, predating the discovery of the ChaC family. Initially considered the sole enzyme capable of degrading GSH, GGT hydrolyzes the γ-glutamyl bond of extracellular reduced and oxidized GSH (22). This results in cleaved glutamate, cysteine and glycine while facilitating the transfer of γ-glutamyl moiety of GSH to either water (hydrolysis) or substrates such as peptides (transpeptidation). Consequently, GGT is classified as a bisubstrate enzyme (23).

GGT is a glycosylated heterodimer protein formed by the non-covalent combination of a heavy chain subunit (relative molecular mass, 50,000-62,000) and a light chain subunit (relative molecular mass 22,000-30,000; Fig. 2) (24). The human GGT family members are synthesized and cleaved by an autocatalytic processing reaction. They have a conserved 'sandwich-like' three-dimensional domain with four layers of αββα folds (23). The catalytic site of GGT consists of two successive regions: Well-characterized donor site, specifying the substrates to which donor γ-glutamyl groups bind, and the acceptor site, about which little is currently known regarding the involved residues (24).

Anchored in the plasma membrane by the N-terminus of the heavy chain across the membrane segment, GGT protein, under physiological conditions, is typically confined to the plasma membrane. It is distributed on the apical surface of epithelial and endothelial cells in glands and lumens. A unique characteristic of GGT is that it is located on the extracellular surface of mammalian cells with a catalytic active site oriented to the extracellular environment (11). The kidney expresses GGT at the highest levels, while notable expression is also found in bile canaliculi of hepatocytes, ducts within the pancreas, the apical surface of the intestinal epithelium and the luminal surface epithelium of many reproductive organs (25).

A GGT gene family exists in the human genome (Table I), suggesting that regulating GGT activity may be associated with activating different GGT genes rather than identifying distinct gene loci (26).

The human genome sequence contains 13 GGT homologs, the most active of which is GGT. The GGT gene was first discovered on human chromosome 22 at q11.1-q11.2 (27), although it was also subsequently discovered on additional autosomes (26). In addition, two homologs, GGTLC1 (previously GGTL6, GGTLA4) and GGTLC2, which may only encode the light-chain portion of GGT, as well as at least three other homologs, exhibit activity: GGT5 (formerly GGL, GGTLA1/GGT-rel), GGT6 (formerly rat GGT6 homologous) and GGT7 (formerly GGTL3, GGT4) (28). While GGT5 and GGT1 share 40% of the amino acid sequence (22), GGT5 is not as active in hydrolyzing GSH, GSSG and leukotriene C4 as GGT1 is (29). Despite the absence of verified protein-coding activity, GGT6 and GGT7 exhibit aberrant expression in conditions such as tumors (30-32) and pancreatic disease (33). Furthermore, proteins expressed by the human GGT2 gene share 94% of the amino acid sequence encoded by GGT1 (34), even though GGT2 only encodes inactive pro-peptides. GGT2 also exhibits abnormal expression in tumors (30) and upregulating GGT2 can overcome H₂O₂-induced apoptosis (35). These findings suggest that GGTs play potential roles in disease physiology and pathology.

As a member of the N-terminal nucleophilic hydrolase superfamily (Ntn), GGT uses a highly conserved catalytic mechanism. An N-terminal Thr residue is essential for substrate priming in human GGT (22), with the substrate binding site featuring a key Thr side chain. This facilitates conversion of the γ-glutamyl bond of GSH into an acyl bond, releasing Cys-Gly and glutamate (11).

While the human GGT gene is not fully characterized, evidence suggests its existence in multiple copies within the human genome (36). As GGT mRNAs share a common coding sequence and have 59 untranslated regions (UTRs), its structural complexity is evident (37). Understanding of the human GGT promoter remains limited (38).

A number of promoters control human GGT transcription, and the resulting transcripts undergo selective splicing in untranslated regions and coding sequences (37). The initiation of GGT mRNA transcription involves cis-reactive elements, including the cis-regulatory element (TRE) and the binding element for activator protein 1 (AP-1) (38). TRE, also known as 12-O-tetracylacylphobolol 13 acetic acid reactive element, incorporates binding sites for activating protein 2 (AP-2) and specific protein 1 (Sp1; Fig. 3).

A study (39) on HeLa cells highlighted that phorbol 12-myric acid 13-acetic acid enhance the expression of human GGT, pinpointing its binding site at the AP-1 binding site 2,214/2,225 nucleotides upstream from the transcription start site. More research is necessary to understand the transcriptional mechanism of the human GGT gene and promoters.

Although rat and human GGT promoters may have similar structures, the human promoter is more complex. As the rat GGT gene is single-copy, replicating unique genes after species transfer between rats and humans likely results in multiple human GGT genes (40). Therefore, rat GGT may provide insight into human GGT expression and regulation.

In rats, GGT expression is controlled by a tandem P1-P5 promoter, facilitated by variable splicing. This yields transcripts sharing the same coding region and diverse 5'-UTRs (38). These unique promoters have high tissue stage-specificity (38).

Upregulation of GGT activity is primarily dependent on the Ras protein and its downstream effectors, which include extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/AKT and c-Jun N-terminal kinase (JNK) signaling pathways (Fig. 3) (41). The Ras protein is a key regulator of signaling pathways key for normal cell proliferation. Malignant phenotype of tumor cells is caused by the Ras gene, often mutated or active in tumor cells, and leads to aberrant tumor cell proliferation, programmed cell death, invasion and angiogenesis (42).

Through the electrophile response element (EpRE), ERK1/2 and p38MAPK signaling pathways actively contribute to upregulation of GGT promoter 5 (GP5) activity in response to 4-hydroxynonenal (HNE) (43). Following activation by redox signaling downstream of the MAPK signal pathway, the EpRE binding protein, also an oxidative stress-associated transcription factor, induces the dissociation of Nrf2 from Keap1 via redox modification and/or phosphorylation of Nrf2. Subsequently, Nrf2 translocates from cytoplasm to the nucleus, forming heterodimers with other proteins to bind to EpRE. This leads to the amplification of GGT transcription. In alveolar type II (L2) cells, EpRE motif in the proximal region of GP5 EpRE induces the expression of the major GGT transcript mRNA V-2 in the lung (38). However, pretreatment with ERK1/2 pathway inhibitor (PD98059) or p38MAPK inhibitor (SB203580) partly decreases the expression of GGT mRNA V-2 induced by HNE in L2 cells (38). Furthermore, activated Ras is also implicated in inducing activation of GP2 and increasing expression of GGT transcriptional products and protein in colon cancer cells treated with naphthoquinone during acute oxidative stress (Fig. 3) (41).

Moreover, inflammatory conditions significantly enhance GGT levels by activating the NF-κB pathway. NF-κB, the core transcription factor in the NF-κB signaling pathway, is a dimer family formed by p50/p105/NF-κB1, p52/p100/NF-κB2, c-Rel, p65/RelA and RelB (44). It regulates the expression of chemokines, cytokines, transcription factors and regulatory proteins, playing a crucial role in inflammation and immunity (44). Upon cell stimulation by an external signal, the NF-κB dimer is released from its inhibitor (IκB) and freely transferred into the nucleus. When the NF-κB pathway is activated, it triggers production of pro-inflammatory proteins downstream, such as tumor necrosis factor-α (TNF-α), which in turn causes inflammatory responses and pain. Furthermore, by serving as NF-κB activators, these inflammatory cytokines intensify inflammation by further triggering the NF-κB pathway (45). In the 536 bp site of the proximal promoter of GGT, a binding site exists between p50, TNF-α and Sp1, regulated by the activation of the NF-κB signaling pathway, thereby promoting expression of GGT and inducing inflammatory response (Fig. 3) (46). Moreover, it has been reported (46) that inhibitors of the NF-κB pathway can effectively block the trans-activation of GGT promoters at different levels. For example, remicade, a clinically used anti-TNF-α antibody targeting the p50 and p65 NF-κB subtype of small interfering RNA and curcumin, a well-characterized natural NF-κB inhibitor that is also a dominant negative inhibitor of κBα (IκBα), can inhibit GGT activation through distinct mechanisms. This suggests the involvement of the NF-κB pathway in regulating GGT expression. Therefore, inflammatory conditions may increase GGT synthesis, potentially acting as a cellular protective mechanism under increased oxidative stress or promoting inflammatory progression. Further research is necessary to explore these possibilities.

In addition to the well-established extracellular GSH degradation mechanism, studies have shown an additional intracellular hydrolysis pathway for GSH degradation (47,48). ChaC protein features a BtrG/γ-GCT fold and distinctive β-barrels surrounded by α-helices (Fig. 4) (13). Mammals exhibit two isoforms of ChaC: Mammalian pro-apoptotic factor ChaC1 (formerly MGC4504) and its homologous counterpart ChaC2. Conversely, only one ChaC member is present in lower eukaryotes, especially in unicellular eukaryotes (47).

The human ChaC1 gene is on chromosome 15q15.1 and comprises three exons, encoding a protein with 222 amino acid residues and a molecular weight of ~25 kDa (49). ChaC1, 30% identical to mammalian and prokaryotic genes, serves a crucial role in basic physiology (12).

ChaC2 is on chromosome 2p16.2 and encodes a protein with 184 amino acid residues and a molecular weight of ~20.9 kDa (20,47,50). A phylogenetic study (47) highlighted that ChaC2 evolved earlier than ChaC1 and shares key structural similarities with ChaC proteins of lower eukaryotes. Human ChaC2 and ChaC1 share 50% of their protein identity (47). ChaC2 typically exists in dimer crystals with a unique flexible loop 2 structure, with an open conformation that can facilitate close contact with crystallographically adjacent ChaC2 molecules. Additionally, ChaC2 E74Q/E83Q active site mutants exhibit a closed conformation, regulating the degradation activity of ChaC2 to GSH (20).

ChaC1, an inducible enzyme, can be expressed under specific stresses or pathological conditions (12,47). It selectively hydrolyzes GSH, producing Cys-Gly and 5-oxoproline (a cyclized form of glutamate) (11,51), thereby accelerating formation of the cellular oxidative environment. The Michaelis constant of ChaC1 for GSH is ~2.2±0.4 mM (47), comparable with the concentration of intracellular GSH (1-10 mM) under physiological conditions. ChaC1 often forms dimers or tetramers, with dimerization being key for regulating enzyme activity and substrate specificity. Under stress or tumor growth, there is an increased need for enzyme breakdown, which leads to formation of dimers or longer oligomers of ChaC1 (20).

ChaC2 is constitutively expressed and exhibits catalytic efficiency for GSH 10-20 times weaker than that of ChaC1 (47). The lower activity of ChaC2 may be partly attributed to flexible loop 2, acting as a gating function to achieve specificity for GSH binding and regulate a constant GSH degradation rate. In addition, the Glu74 and Glu83 residues of ChaC2 are key for directing the conformation of the enzyme and regulating enzyme activity (20).

Various signals, including endoplasmic reticulum (ER) and oxidative stress and viral infection, activate ChaC1 promoters in different cell types, cellular processes and diseases through the unfolded protein response (UPR) (12,48,52). ChaC1 is downstream of the protein kinase R-like ER kinase (PERK)/eukaryotic initiation factor-2α (eIF2α)/activating transcription factor (ATF) 4/ATF3/CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) pathway, serving as a pro-apoptotic and pro-ferroptosis component downstream of UPR. ATF4, ATF3, CHOP and C/EBP-β upregulate ChaC1 transcription (Fig. 5). The activation of ChaC1 by UPR primarily relies on ATF4, while the involvement of CHOP, C/EBP-β, and ATF3 is indirect (12). A direct relationship between ATF4 and regulatory elements within the ChaC1 promoter has been identified. A-267 ATF/cAMP response element (CRE) in conjunction with a novel-248 ATF/CRE modifier (ACM), serving as a binding site for ATF4 and ATF3 transcription factors, regulates the activity of the basic ChaC1 promoter. Among these elements, ATF3 predominantly regulates the basal and stress-induced expression of ChaC1 through ATF/CRE, while ATF4 primarily regulates stress-induced ChaC1 expression through ATF/CRE and ACM (48). Additionally, conserved-209 CEBP-ATF response element has a limited impact on regulating human ChaC1 transcription (48,53).

Amino acid starvation can induce the expression of ChaC1 (Fig. 5). The amino acid starvation response activates ATF4 via the general control nonderepressible 2 (GCN2)/eIF2a/ATF4/ATF3 pathway (54). Both ER stress and amino acid starvation induce stress synergistically by activating ATF4 and ChaC1 is one of the downstream targets of ATF4. In regulating ChaC1 expression, C/EBP-β has been observed to recruit ATF4 to the ChaC1 promoter in response to ER stress (48). However, the precise C/EBP-β response element on the ChaC1 promoter remains unclear (48). Further studies are necessary to elucidate the detailed mechanism of C/EBP-β-mediated ATF4 recruitment and its impact on ChaC1 expression. These findings highlight the intricate nature of ChaC1 transcriptional regulation and underscore the importance of maintaining appropriate redox balance in cells (48). The mechanism governing ChaC1 protein expression requires further characterization.

Understanding of the regulation mechanism of ChaC2 is limited. A previous study (47) suggested that ChaC2 is expressed at higher basal levels under physiological conditions than ChaC1. However, under cellular stress such as ER stress or amino acid starvation, ChaC1 is upregulated, while ChaC2 expression remains unaffected (47). Thus, ChaC2 serves as a constitutively expressed protein for basal hydrolysis of GSH, acting as a steward for slow and continuous GSH turnover (47).

GSH-degrading enzymes key central to coordinating cellular metabolism by regulating amino acid availability under physiological conditions (11). The modulation of the cellular stress environment relies on regulation of GSH metabolism (4). Tumor cells, in their quest for survival and proliferation, generate abnormally high levels of oxidative stress, partly due to increased cellular redox buffer GSH (87). Therefore, the role of GSH-degrading enzymes is key to regulate the cell stress environment.

The GSH degradation pathway initiated by extracellular GGT effectively controls the intracellular oxidative stress environment (38). GGT deficiency leads to oxidative stress and cellular vulnerability to oxidative damage. Animal studies (88-90) have indicated that GGT knockout mice exhibit 20% of the plasma cysteine concentration in wild-type mice. GGT knockout mice experience increased accumulation of DNA oxidative damage, decreased intracellular GSH levels, elevated oxidative stress and death by 10 weeks of age due to cysteine deficiency (88). Patients with partial GGT homozygous deletions report glutathionuria and neurodevelopmental disorder (91). Hence, maintaining regular GGT expression is essential for cellular GSH homeostasis and protecting cells against oxidative stress.

Cells overexpressing GGT gain an advantage in environments with physiological and limited cysteine concentrations by efficiently utilizing extracellular GSH as a source of cysteine (92). Tumor cells with elevated intracellular GSH levels often induce overexpression of GGT. It has been reported (22) that both GSH depletion and GGT inhibition significantly enhance cytotoxicity under oxidative stress in tumor cells. Tumor cells with high GGT expression demonstrate notable oxidative stress tolerance without DNA damage, while clones with low GGT expression exhibit increased sensitivity to oxidative stress and apoptosis (93). As a marker of oxidative stress, GGT expression in advanced tumor cells surpasses that in early tumor cells. The increased oxidative stress and impaired immune responses may be key for promoting cancer progression to advanced stages and may be induced by inflammatory mediators within the tumor (94). Therefore, upregulation of GGT in tumor cells provides a potential mechanism to resist oxidative stress and foster tumor progression.

Simultaneously, changes in the tumor microenvironment generate persistent ER stress signals in various types of tumor (95) such as colorectal (96), pancreatic cancer (97) and so on. This state has a dual effect on tumor cells, on one hand controlling several tumor-promoting features, on the other hand dynamically reprogramming immune cells and inducing tumor cells autophagy, apoptosis and ferroptosis (95). ChaC1, a component of the UPR pathway, is a target of ferroptosis induced by ER stress signals (12). Thus, ChaC1 is as a key regulator of tumor development, metastasis and responses to chemotherapy, targeted therapy and immunotherapy.

Previous research (78) has also revealed a broader range of functions for ChaC2 than previously understood. ChaC2 inhibits ChaC1-mediated GSH degradation, indicating competition with ChaC1 to maintain GSH homeostasis (78). ChaC2 directly regulates GSH production via a ChaC1-independent pathway (78). ChaC2 enhances GSH production by upregulating Nrf2, a key regulator of antioxidation, and its downstream glutamate-cysteine ligase (78). These diverse actions underscore the importance of ChaC2 in maintaining cellular redox homeostasis and antioxidation mechanisms.

Extensive crosstalk exists between oxidative and ER stress as oxidative stress can disrupt redox homeostasis in the ER, triggering ER stress (95). GGT and ChaC1 may be key factors in this mechanism. A study (98) demonstrated that GGT1 and GGT7 stimulate induction of ER stress-related protein, CHOP-10 and immunoglobulin heavy chain binding protein BiP, indicating specific roles for these GGT protein subtypes in ER stress response. This suggests possible crosstalk between GGTDelta1, GGTDelta7 and ChaC1 via the ER stress/CHOP pathway. A recent study (99) proposed that the ATF4/CHOP/ChaC1 signaling pathway might be vital for apoptosis induced by crosstalk between oxidative and ER stress. Under extreme heat stress, cells produce a large amount of ROS, leading to oxidative stress and protein misfolding in the ER, resulting in ER stress and triggering ChaC1-associated UPR. Moreover, induction of ChaC1 serves an essential regulatory role in ER stress-mediated apoptosis of cancer cells induced by the anticancer monosaccharide xylitol, leading to secondary induction of oxidative stress in treated cells and apoptosis (100). This evidence collectively demonstrates the key role of GGT and ChaC1 in mediating crosstalk between oxidative and ER stress.

Programmed cell death, encompassing apoptosis, ferroptosis, necrotic apoptosis and autophagy, is instigated by a series of intracellular processes (101). GGT- and ChaC1/ChaC2-mediated intracellular GSH depletion can concurrently or sequentially initiate multiple forms of programmed cell death. Studies (101,102) have indicated that the GSH/GSSG redox status serves as a vital indicator of tumor programmed cell death, consistently associating programmed cell death with a decrease in the GSH/GSSG ratio. Therefore, targeting GGT and ChaC1/ChaC2 to modulate programmed cell death holds implications for tumor therapy.

Apoptosis, the quintessential programmed cell death process, primarily relies on the caspase family for initiation and is typically characterized by membrane contraction, chromatin concentration and apoptotic body formation (103). In tumor cells, the apoptosis pathway is often impeded by various mechanisms, many of which contribute to intrinsic resistance to chemotherapy, the most prevalent anticancer therapy (104). Reducing GSH impairs cellular antioxidant regulation, increasing ROS production, thereby accelerating mitochondrial damage and apoptosis induction (101). Consequently, inhibiting GGT1 in tumor cells facilitates induction of apoptotic phenotypes (105). Simultaneously, research (106) on another member of the GGT family has shown that low GGT7 expression may elevate cell ROS levels, inhibiting apoptosis and fostering tumor proliferation. This suggests variations in regulation of ROS levels within the GGT family. ChaC1 overexpression can augment apoptosis by activating caspase-3/9, degradation of poly (ADP ribose) polymerase, induction of autophagy, ROS generation, increased intracellular calcium and loss of mitochondrial membrane potential (69).

Ferroptosis, a distinct iron-dependent form of cell death, arises from lethal accumulation of lipid peroxides (107). In tumor cells, evasion of ferroptosis mediated by oncogenes or carcinogenic signaling contributes to tumor onset, progression, metastasis and resistance to treatment. Simultaneously, some tumor cells, owing to specific mutations, elevated ROS levels and other unique biological features, exhibit ferroptosis susceptibility, with their survival hinging on the ferroptosis defense system (108). For example, ferroptosis resistance is conferred by frequently occurring PI3K activating mutations or loss of phosphatase and tensin homolog deleted on chromosome 10 function in human cancer such as lung adenocarcinoma (109) and breast cancer (110) and so on. Conversely, inhibition of the PI3K/AKT/mTOR signaling axis sensitizes cancer cells to ferroptosis induction (111). Consequently, targeting ferroptosis regulation holds implications for cancer immunotherapy and tumor suppression.

Regarding ferroptosis regulation, GGT-activated extracellular GSH catabolism produces iron-derived ROS, inducing lipid peroxidation via NF-κB pathway activation (112). GGT-mediated GSH catabolism via lipid peroxidation enhances NF-κB DNA binding capacity in tumor cells (113). GGT increased intracellular GSH levels (114), restores the reduced GSH/GSSG ratio and reactivates GSH peroxidase 4, a core ferroptosis regulator. Therefore, elevated GGT expression increases tumor cell resistance to ferroptosis, safeguarding cells from ROS and lipid peroxidation, thus driving tumor cell proliferation, metastasis and chemotherapy drug resistance (85,107). In addition, GGT activates the mTORC1 pathway and inhibits integrated stress response (ISR) by modulating cystine-GSH crosstalk. This inhibition of ferroptosis promotes cancer development and other cysteine-deficient diseases (115). Inhibiting GGT impairs GSH ability to restore mTORC1 signaling and ISR, inducing ferroptosis. This implies that the role of GGT in inducing capacity of GSH to release cysteine, rather than GSH itself, modulates the mTORC1 pathway, ISR and ferroptosis. By contrast, ChaC1 induces ferroptosis in tumor cells by activating the GCN2/EIF2α/ATF4 pathway to intensify cystine depletion (116). ChaC1 overexpression depletes GSH, initiating and executing ferroptosis. The deletion of ATF4, an upstream factor of ChaC1, in embryonic fibroblasts, results in a ferric oxide-dependent death phenotype, emphasizing the role of ATF4 as a downstream molecule of the eIF2α/ATF4 pathway (117). Hence, ferroptosis control is associated with ChaC1 expression.

Necroptosis, a regulated form of cell death primarily dependent on receptor-interacting protein kinase 3 and mixed lineage kinase domain-like, is characterized by widespread cytoplasm and organelle swelling, plasma membrane rupture and release of cell components into the microenvironment (118). This pro-inflammatory form of cell death holds implication for combating pathogen infection, inflammatory progression (119), and therefore may also contribute to early prevention of inflammatory cancer transformation.

Understanding of the impact of GGT and ChaC1/ChaC2 on necroptosis is limited. GGT, one of the virulence factors of Helicobacter pylori, has been demonstrated to induce necroptosis in gastric epithelial cells (119). In the early stage of infection, necroptosis may serve a protective role in the mucosa by triggering an immune response (119). However, as the disease progresses, uncontrolled necroptosis exacerbates mucosal inflammation and contributes to the transformation of inflammation into cancer (119). Additionally, ChaC1, by stimulating the GCN2/eIF2α/ATF4 pathway, enhances necroptosis induced by cystine deprivation (54).

Autophagy, a highly conserved cellular degradation process, involves breaking down cytoplasmic components and damaged organelles via lysosomes, recycling resulting macromolecules to shield cells from diverse stressful conditions. Traditionally viewed as a cytoprotective mechanism, autophagy, when excessive, can also instigate cell death and contribute to tumor suppression (120). GSH is implicated in inducing autophagy, where low GSH levels serve as a signal activating autophagy as an adaptive stress response (101). Inhibition of GGT (105) and elevated expression of ChaC1/ChaC2 (69,79) are associated with autophagy phenotype. Nevertheless, evidence (79,121) elucidating the precise mechanisms and interactions between GGT and ChaC1/ChaC2 and initiation and promotion of autophagy remains limited.

Chronic non-specific inflammation is pivotal in tumorigenesis (122) and is a primary environmental factor contributing to the onset and metastasis of specific types of cancer such as non-small cell lung cancer and colorectal cancer (122,123). Various blood tests, either individually or in combination, reflecting local or systemic inflammation, are valuable prognostic indicators for multiple tumor types (124).

GGT serves as a well-established inflammatory marker associated with inflammatory environments and malignancy (125,126). Combining serum albumin and GGT levels serves as an inflammatory indicator for assessing the prognosis of hepatocellular carcinoma (HCC). Patients with elevated GGT and decreased albumin expression exhibit poorer prognosis, revealing significant differences in tumor characteristics, including larger maximum tumor diameters, more tumor nodules and potential for macroscopic vascular invasion and higher serum tumor marker levels (124).

Furthermore, although there is limited research on the effect of human GGT on the colonization of H. pylori, it is known that H. pylori GGT is a bacterial virulence factor that contributes to the colonization of H. pylori in human stomach parietal cells, hence inducing inflammation and gastric parietal cell carcinogenesis (127). In H. pylori infection, stimulation of H. pylori GGT accelerates the decrease of GSH levels in gastric epithelial cells, thereby exacerbating ROS production, leading to DNA damage and playing a key role in the emergence of chronic gastritis and gastric cancer (127). H. pylori GGT is also key for the tolerogenic effect of dendritic cells in H. pylori infection, ensuring bacterial persistence and cross-protection from chronic inflammation and autoimmune diseases by promoting H. pylori to reprogram dendritic cells into tolerogenic phenotypes (128). More research is needed to determine whether human GGT functions similarly in H. pylori-infected gastric parietal cells.

Human ChaC1 has potential ability to promote inflammation (129). ChaC1 is highly expressed in gastric cancer associated with H. pylori infection (129). Infection with H. pylori triggers ChaC1 overexpression in gastric epithelial cells, leading to GSH degradation and ROS accumulation, suppressing nucleotide alterations in TP53 that induce tumor suppressor gene expression (130). Overexpression of ChaC1 in H. pylori-infected parietal cells may also lead to H. pylori-induced somatic mutation, thereby promoting the development of gastric cancer (131). In summary, high expression of human ChaC1 is involved in inducing development of gastric cancer.

ChaC1 expression varies in normal and cystic fibrosis bronchial epithelial cells, with low ChaC1 expression hypothesized to play a significant role in regulating the chronic inflammatory response induced by Pseudomonas aeruginosa (Pa) (132). When exposed to Pa and its virulence components, normal bronchial epithelial cells preferentially produce ChaC1. Conversely, low ChaC1 expression is associated with increased secretion of inflammatory markers interleukin-8, interleukin-6 and prostaglandin E2 in the presence of lipopolysaccharide and flagellin stimulation (132). Low ChaC1 expression also promotes increased phosphorylation of NF-κB p65, possibly contributing to the exacerbation of characteristic inflammation in the lungs of patients with cystic fibrosis following Pa infection (132). Cystic fibrosis itself is a risk factor for various cancers, including lung cancer (133).

High GGT and low ChaC1 expression in cancer cells are pivotal factors in developing drug-resistant phenotypes in tumors (86). GGT expression provides cells with an additional supply of cysteine, while low ChaC1 expression hinders degradation of intracellular GSH. Both factors contribute to GSH consumption in tumor cells during anticancer chemotherapy, leading to drug resistance. Maintaining high intracellular GSH expression preserves redox status, allowing cells to respond to proliferative and differentiation signals in tissue after toxin injury (22) and rapidly supplement GSH during pro-oxidant anticancer therapy.

Chemotherapy-resistant tumors often exhibit high GGT expression, exemplified by cisplatin resistance (134). Platinum (II) class antitumor drugs such as cisplatin and oxaliplatin, widely used in cancer chemotherapy, target DNA damage and overall cytotoxicity in tumor cells, resulting in cell death (135). GGT-related detoxification of Pt(II) medication is a key mechanism of drug resistance (136). Cisplatin can strongly bind to mercaptan metabolites produced by GGT-mediated GSH cleavage, reducing Pt ion entry into cells and inactivating Pt drugs outside the cell (136). GGT-transfected cells show decreased DNA platinization, resulting in decreased sensitivity to cisplatin and lower susceptibility to DNA damage (114). GGT-transfected HeLa cells exposed to cisplatin exhibit a 10-fold increase in cisplatin resistance (134). Systematic inhibition of GGT expression is conducive to suppressing nephrotoxic side effects of cisplatin without diminishing its intracellular toxicity to tumor cells (137). These findings underscore the key role of high GGT expression in promoting drug-resistant phenotypes in tumor cells.

By contrast with GGT, high ChaC1 expression, combined with drugs such as bortezomib and docetaxel, inhibits tumor cell viability by blocking cell cycle progression from the G1 phase to the mitotic S phase. Additionally, high ChaC1 expression increases tumor cell sensitivity to anti-tumor drugs by inducing ER stress and ferroptosis (65,69,138). GSH depletion triggered by the Nrf2/ATF3/4/ChaC1 pathway appears to be the primary factor inducing death in drug-resistant tumor cells (138), positioning ChaC1 as a key target for certain potential anti-tumor drugs such as busatol (139) and glaucocalyxin A (140). Low expression of ChaC1 appears in the drug-resistant phenotype of tumor cells (141), which may be related to enhanced intracellular GSH levels.

Early tumor screening and diagnosis are key for effective treatment and favorable prognosis. Reliable and specific tumor biomarkers are key for accurate screening and diagnosis. The aberrant expression of GSH-degrading enzymes is associated with the prognosis of certain cancers such as gastric adenocarcinoma (72), breast cancer (74) and so on, making them promising tumor biomarkers.

GGT has been extensively studied and is widely used as a biomarker in clinical practice (142-144). Serum GGT activity is a rapid, reliable and cost-effective method to assess liver function (145). Therefore, GGT has the potential to serve as a tumor biomarker. Elevated GGT expression can indicate the early-stage risk of tumor development, as suggested by an epidemiological study linking high GGT expression with increased risk of prostate cancer development (145). Moreover, high GGT expression indicates poor prognosis in various types of tumors, including renal cell carcinoma and prostate and urothelial cancer (145). The Cancer antigen 19-9/GGT ratio serves as an independent prognostic predictor following radical resection in ampulla carcinoma (146). GGT6 and GGT2 are novel synergistic prognostic biomarkers for low-grade glioma and GBM, potentially aiding early detection (30).

GGT probes have been developed to detect GGT activity accurately for tumor imaging. However, the imaging process often requires organic solvents, posing risks of damage to the enzyme and body. A water-soluble fluorescent probe, TCF-GGT (147), has shown promise by producing red fluorescence during GGT catalytic hydrolysis without interference from the background. This water-soluble compound holds clinical value due to its practical imaging, quick metabolic cycle and excellent water solubility. Due to shallow tissue penetration of many GGT-targeted fluorescent probes, their clinical application is limited. A novel positron emission tomography imaging probe, ([¹⁸F]GCPA)₂, has been designed to sensitively and precisely monitor GGT levels in living subjects because of the high sensitivity and intense tissue penetration of positron emission tomography (148). In addition, Cy-GSH, a zero-crosstalk ratio near-infrared GGT fluorescent probe, has also been designed to visualize deep cancer in vivo. The probe accurately visualizes tumors and metastases in mice, suggesting that it could be a convenient tool for fluorescence-guided cancer surgery (149). More clinically applicable GGT-targeted probes for visualizing deep cancer need further studies, which may contribute to the early diagnosis of clinical tumors.

ChaC1, a GSH-degrading enzyme, is biomarker for certain types of tumors. In vitro, ChaC1 induces cell death in KIRC cell lines, signifying its potential as an effective marker for poor prognosis in KIRC (70). High ChaC1 expression also serves as a biomarker for adverse outcomes in gastric adenocarcinoma (72), corpus endometrial (73) and breast cancer (74) and uveal melanoma (75). Notably, a positive correlation exists between ChaC1 expression and immune infiltrating cells in corpus endometrial carcinoma (73). In uveal melanoma (75,150), ChaC1 is associated with poor overall, progression-free and disease-specific survival and progression-free interval, making it a promising indicator of unfavorable tumor prognosis. In aggressive breast tumor subtypes such as triple-negative breast cancer, ChaC1 exhibits significant upregulation (151). In addition, malignant breast cancer tissue with active lymph node metastases and high proliferation rates demonstrates elevated levels of ChaC1, supporting its potential for defining tumor progression and metastasis (Table II).

The abnormal expression of ChaC2 as a tumor biomarker has garnered recent attention (50,81). As an independent marker of poor prognosis for certain types of tumors such as breast cancer (50) and hepatocellular carcinoma (82), ChaC2 can monitor early tumor occurrence and treatment effects. Low expression of ChaC2 independently indicates poor prognosis in gastrointestinal tumors. ChaC2 induces mitochondrial apoptosis and autophagy via UPR, serving a pivotal role as a tumor suppressor gene in the onset, proliferation and metastasis of gastric and colorectal cancer (79). Immunohistochemistry and western blot analysis reveal ChaC2 downregulation in most tumor tissue, with ChaC2 expression positively correlating with 3-year survival rate (79). However, recent findings indicate that high ChaC2 expression is inversely correlated with overall survival in patients with breast cancer (50). Elevated ChaC2 expression is also associated with poor prognosis in HCC (82). This underscores the tissue-specific effect of ChaC2 on tumors, necessitating further study (Table II).

Further, the previous study (152) integrates traditional predictors and ChaC1, a novel biomarker, to develop a prognostic score for patients with tumors. The score can be used as a reference for clinical chemotherapy decision-making (152). In evaluating patients with primary breast cancer, including ChaC1 mRNA expression levels in the scoring model led to changes in chemotherapy decisions in 16% of patients (152). In addition, ChaC1 has been included in prognostic models of renal cell carcinoma (153) and glioblastoma (154). Including ChaC1 in tumor prognosis predictions may guide personalized treatment options. ChaC1/ChaC2 may be promising targets for precision medicine.

While studies (50,73,74,151,82) have reported ChaC1/ChaC2 as tumor biomarkers, the development of ChaC1/ChaC2 tracer fluorescent probes remains unexplored. Fluorescent probes are the foundation for tumor-specific imaging in clinical applications. Therefore, developing optical probes to track ChaC1/ChaC2 in vivo or in vitro is key for fully realizing the clinical potential of ChaC1/ChaC2 as tumor markers.

Drug selection and emerging drug development. Exploration of GSH-degrading enzyme modulators presents a promising avenue for impeding cancer progression, overcoming tumor resistance to pro-oxidative therapy and preserving tumor sensitivity to chemotherapy (Table III).

Diminishing GGT expression is beneficial for tumor treatment. Classical GGT inhibitors, including glutamate analogs such as acivicin, 6-diazo-5-oxo-L-norleucine, and azaserine (155), have shown clinical toxicity against embryonic cells (156). γ phosphonoglutamate analogs such as GGsTop and derivatives of the lead compound N-[5-(4-methoxybenzyl)-1,3,4-thiadiazol-2-yl]benzenesulfonamide, OU749, represent another class of GGT inhibitors (22). GGsTop targets GGT, can reduce the immunosuppressive function of anti-tumor drugs when used in combination with anti-tumor drugs (157). OU749, a species-specific uncompetitive GGT inhibitor, exhibits low toxicity and a broad therapeutic window for humans.

The development of ChaC1 modulators introduces innovative approaches to cancer treatment. Various potential ChaC1 modulators have been reported, targeting tumor cell cycle arrest and apoptosis (68,158). Metformin, by regulating the Loc100506691-miR-26a-5p-miR-330-5p-CHAC1 axis, induces cell cycle arrest in the G2/M phase, inhibiting cancer cell proliferation (71). Nisin, an apoptotic bacteriocin, induces ChaC1 activation, calcium influx and cell cycle arrest in the G2 phase, leading to increased apoptosis and decreased cell proliferation in vitro and in vivo (68). Atovaquone, an anti-parasitic agent, induces eIF2a phosphorylation at serine 51, amplifying the eIF2α/ATF/CHOP/ChaC1 signaling pathway under ER stress (158).

Natural extracts and their bioactive compounds have recently gained recognition as potential lead molecules in drug discovery for cancer treatment (159,160). They offer an alternative to chemical synthetic drugs, potentially minimizing toxic side effects and holding promise for enhancing clinical anti-tumor therapy. GGT and ChaC1 modulators derived from natural extracts have been explored. Ovothiol, a marine-derived 5(N)-methyl-thiohistidine, is a more effective non-competitive-like GGT inhibitor than traditional counterparts such as acivicin, 6-diazo-5-oxo-norleucine and azaserine (105,161). Ovothiol (105) induces apoptosis and autophagy in GGT-overexpressing cells. The anticancer monosaccharide xylitol (100) and natural Chinese herbal extracts, including dihydroartemisinin (66), artesunate (67), glaucocalyxin A (140), and tanshinone IIA (162), upregulate Prostaglandin-Endoperoxide Synthase 2, p53 and ChaC1 expression. This amplifies the ATF4/CHOP/ChaC1 cascade under ER stress, resulting in decreased intracellular GSH and cysteine, increased intracellular ROS, redox homeostasis disruption, secondary oxidative stress in cancer cells and selective ferroptosis of cancer cells (66,100).

In clinical treatment of tumors, a key goal is to decrease toxicity and resistance of anti-tumor drugs by regulating low expression of GGT (22) and high expression of ChaC1 (65). Blocking nephrotoxicity induced by the anti-tumor drug cisplatin is achieved by inhibiting GGT (137). The unique renal sulfhydryl acid metabolic pathway, involving degradation of GSH by GGT, contributes to cisplatin nephrotoxicity (163). Supramolecular Pt prodrug nano-assemblies inhibiting c-glutamyl transferase prove beneficial for overcoming tumor resistance (136). GGT inhibitor amlodipine, through its anti-inflammatory effect, suppresses p38 MAPK-triggered pro-inflammatory signaling, decreasing expression of TNF-α and other downstream targets while upregulating expression of the transcription factor Nrf2 and the antioxidant protein heme oxygenase-1 (164). Amlodipine diminishes the pro-apoptotic effector/anti-apoptotic protein expression ratio induced by cisplatin, preventing inflammation, oxidative stress and apoptotic damage (165).

Combining ChaC1 overexpression with chemotherapy is advantageous in decreasing cell drug resistance. Temozolomide, for example, induces binding of ChaC1 to the Notch3 protein, inhibiting activation of Notch3. This weakens the Notch3-mediated downstream signaling pathway, inducing glioma cell death and indicating that ChaC1 can influence the cytotoxicity of tumor cells induced by temozolomide (69). The omega-3 fatty acids docosahexaenoic acid and eicosapentaenoic acid serve a role in reducing the resistance of tumor cells to bortezomib by activating the serine synthesis pathway, mitochondrial folate cycle, methionine cycle-associated GSH synthesis and ChaC1-mediated GSH degradation in tumor cells, ultimately promoting GSH degradation (138).