Glutathione­degrading enzymes in the complex landscape of tumors (Review).

Glutathione (GSH)­degrading enzymes are essential for starting the first stages of GSH degradation. These enzymes include extracellular γ­glutamyl transpeptidase (GGT) and intracellular GSH­specific γ­glutamylcyclotransferase 1 (ChaC1) and 2. These enzymes are essential for cellular activities, such as immune response, differentiation, proliferation, homeostasis regulation and programmed cell death. Tumor tissue frequently exhibits abnormal expression of GSH­degrading enzymes, which has a key impact on the development and spread of malignancies. The present review summarizes gene and protein structure, catalytic activity and regulation of GSH­degrading enzymes, their vital roles in tumor development (including regulation of oxidative and endoplasmic reticulum stress, control of programmed cell death, promotion of inflammation and tumorigenesis and modulation of drug resistance in tumor cells) and potential role as diagnostic biomarkers and therapeutic targets.

Identified γ-glutamyl cyclotransferase (GGCT) as a novel regulator in the progression and immunotherapy of pancreatic ductal adenocarcinoma through multi-omics analysis and experiments.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is renowned for its formidable and lethal nature, earning it a notorious reputation among malignant tumors. Due to its challenging early diagnosis, high malignancy, and resistance to chemotherapy drugs, the treatment of pancreatic cancer has long been exceedingly difficult in the realm of oncology. γ-Glutamyl cyclotransferase (GGCT), a vital enzyme in glutathione metabolism, has been implicated in the proliferation and progression of several tumor types, while the biological function of GGCT in pancreatic ductal adenocarcinoma remains unknown. METHODS: The expression profile of GGCT was validated through western blotting, immunohistochemistry, and RT-qPCR in both pancreatic cancer tissue samples and cell lines. Functional enrichment analyses including GSVA, ssGSEA, GO, and KEGG were conducted to explore the biological role of GGCT. Additionally, CCK8, Edu, colony formation, migration, and invasion assays were employed to evaluate the impact of GGCT on the proliferation and migration abilities of pancreatic cancer cells. Furthermore, the LASSO machine learning algorithm was utilized to develop a prognostic model associated with GGCT. RESULTS: Our study revealed heightened expression of GGCT in pancreatic cancer tissues and cells, suggesting an association with poorer patient prognosis. Additionally, we explored the immunomodulatory effects of GGCT in both pan-cancer and pancreatic cancer contexts, found that GGCT may be associated with immunosuppressive regulation in various types of tumors. Specifically, in patients with high expression of GGCT in pancreatic cancer, there is a reduction in the infiltration of various immune cells, leading to poorer responsiveness to immunotherapy and worse survival rates. In vivo and in vitro assays indicate that downregulation of GGCT markedly suppresses the proliferation and metastasis of pancreatic cancer cells. Moreover, this inhibitory effect appears to be linked to the regulation of GGCT on c-Myc. A prognostic model was constructed based on genes derived from GGCT, demonstrating robust predictive ability for favorable survival prognosis and response to immunotherapy.

The γ-glutamyl cycle serves as an amino acids supply system in colorectal cancer organoids under chronic hypoxia.

Malignant tumors are characterized by a hypoxic microenvironment, and metabolic reprogramming is necessary to ensure energy production and oxidative stress resistance. Although the microenvironmental properties of tumors vary under acute and chronic hypoxia, studies on chronic hypoxia-induced metabolic changes are limited. In the present study, we performed a comprehensive metabolic analysis in a chronic hypoxia model using colorectal cancer (CRC) organoids, and identified an amino acid supply system through the γ-glutamyl cycle, a glutathione recycling pathway. We analyzed the metabolic changes caused by hypoxia over time and observed that chronic hypoxia resulted in an increase in 5-oxoproline and a decrease in oxidized glutathione (GSSG) compared to acute hypoxia. These findings suggest that chronic hypoxia induces metabolic changes in the γ-glutamyl cycle. Moreover, inhibition of the γ-glutamyl cycle via γ-glutamyl cyclotransferase (GGCT) and γ-glutamyl transferase 1 (GGT1) knockdown significantly reversed chronic hypoxia-induced upregulation of 5-oxoproline and several amino acids. Notably, GGT1 knockdown downregulated the intracellular levels of γ-glutamyl amino acids. Conclusively, these results indicate that the γ-glutamyl cycle serves as an amino acid supply system in CRC under chronic hypoxia, which provides fresh insight into cancer metabolism under chronic hypoxia.

Genome-wide identification and analysis of the GGCT gene family in wheat.

BACKGROUND: γ-glutamylcyclotransferase (GGCT), an enzyme to maintain glutathione homeostasis, plays a vital role in the response to plant growth and development as well as the adaptation to various stresses. Although the GGCT gene family analysis has been conducted in Arabidopsis and rice, the family genes have not yet been well identified and analyzed at the genome-wide level in wheat (Triticum aestivum L.). RESULTS: In the present study, 20 TaGGCT genes were identified in the wheat genome and widely distributed on chromosomes 2A, 2B, 2D, 3A, 4A, 5A, 5B, 5D, 6A, 6B, 6D, 7A, 7B, and 7D. Phylogenetic and structural analyses showed that these TaGGCT genes could be classified into three subfamilies: ChaC, GGGACT, and GGCT-PS. They exhibited similar motif compositions and distribution patterns in the same subgroup. Gene duplication analysis suggested that the expansion of TaGGCT family genes was facilitated by segmental duplications and tandem repeats in the wheat evolutionary events. Identification of diverse cis-acting response elements in TaGGCT promoters indicated their potential fundamental roles in response to plant development and abiotic stresses. The analysis of transcriptome data combined with RT-qPCR results revealed that the TaGGCTs genes exhibited ubiquitous expression across plant organs, with highly expressed in roots, stems, and developing grains. Most TaGGCT genes were up-regulated after 6 h under 20% PEG6000 and ABA treatments. Association analysis revealed that two haplotypes of TaGGCT20 gene displayed significantly different Thousand-kernel weight (TKW), Kernel length (KL), and Kernel width (KW) in wheat. The geographical and annual distribution of the two haplotypes of TaGGCT20 gene further revealed that the frequency of the favorable haplotype TaGGCT20-Hap-I was positively selected in the historical breeding process of wheat. CONCLUSION: This study investigated the genome-wide identification, structure, evolution, and expression analysis of TaGGCT genes in wheat. The motifs of TaGGCTs were highly conserved throughout the evolutionary history of wheat. Most TaGGCT genes were highly expressed in roots, stems, and developing grains, and involved in the response to drought stresses. Two haplotypes were developed in the TaGGCT20 gene, where TaGGCT20-Hap-I, as a favorable haplotype, was significantly associated with higher TKW, KL, and KW in wheat, suggesting that the haplotype is used as a function marker for the selection in grain yield in wheat breeding.

Signaling pathways upregulating glutathione­specific γ­glutamylcyclotransferase 1 by 3­(5'­hydroxymethyl­2'­furyl)­1­benzylindazole.

Glutathione­specific γ­glutamylcyclotransferase 1 (CHAC1), is an unfolded protein response­induced gene. Although it has been previously reported that CHAC1 transcription is regulated by activating transcription factor (ATF) 4, ATF3 and CCAAT/enhancer­binding protein ß (C/EBPß), the signaling pathways that regulate CHAC1 are largely unknown. It was revealed that 3­(5'­hydroxymethyl­2'­furyl)­1­benzylindazole (YC­1; PubChem ID: 5712), a nitric oxide­independent activator of soluble guanylyl cyclase (sGC), increases CHAC1 levels in cultured human kidney proximal tubular cells (HK­2). Therefore, in the present study, the signaling pathways that induce CHAC1 by YC­1 were investigated in HK­2 cells. YC­1 induced CHAC1 expression in a dose­ and time­dependent manner. KT5823, an inhibitor of cGMP­dependent protein kinase (PKG), partially inhibited CHAC1 upregulation, indicating that the sGC­cGMP­PKG pathway participates in CHAC1 regulation. These results also suggested that other signaling pathways are involved in the regulation of CHAC1. Since antibody array analysis showed the activation of p38, mTOR and Akt, the involvement of these factors was further investigated. Although LY294002 and KU0063794 (inhibitors of Akt and mTOR, respectively) inhibited YC­1­induced CHAC1 expression, SB203580 (an inhibitor of p38) did not. These results indicated that CHAC1 is regulated by the Akt­mTOR pathway. In addition, YC­1 induced endoplasmic reticulum (ER) stress, a regulator of CHAC1 induction. These findings suggested that CHAC1 is regulated by YC­1 through the sGC­cGMP­PKG, Akt­mTOR and ER stress pathways. The present study demonstrated that CHAC1 induction reduced the intracellular glutathione concentration, indicating that CHAC1 plays an important role in intracellular redox homeostasis in tubular cells.

CHAC1 promotes cell ferroptosis and enhances radiation sensitivity in thyroid carcinoma.

ChaC glutathione-specific γ-glutamylcyclotransferase 1 (CHAC1) is involved in intracellular glutathione depletion, ferroptosis, and tumorigenesis. The functional role of CHAC1 expression in thyroid carcinoma has not yet been established. The present study aimed to investigate the impact and mechanisms of CHAC1 on ferroptosis and radiation sensitivity in thyroid carcinoma. CHAC1 expression was examined in tumor tissue specimens and microarrays and thyroid carcinoma cell lines. CHAC1 was silenced or overexpressed by lentivirus transfection in thyroid carcinoma cells. Cell viability and lipid ROS levels were evaluated by Cell Counting Kit-8 and flow cytometry, respectively. The effect of CHAC1 on tumor growth in vivo was also measured. Ferroptosis-related proteins were measured by western blotting. CHAC1 expression was decreased in patients with thyroid carcinoma, and overexpression of CHAC1 suppressed cell viability of BCPAP cells and tumor growth in xenografted nude mice. Exposure to Ferrostatin-1, a ferroptosis inhibitor, significantly attenuated the inhibitory effects of CHAC1 overexpression on cell viability. In CHAC1-overexpressing BCPAP cells, ferroptosis was induced as indicated by increased lipid ROS production and PTGS2 expression. Knocking down of CHAC1 in K1 cells significantly induced cell viability, reduced lipid ROS production and PTGS2 expression, and enhanced GPX4 expression. Such effects were attenuated by RSL3, a ferroptosis inducer. Furthermore, we showed that CHAC1 overexpression enhanced radiation sensitivity in BCPAP cells as indicated by decreased cell viability, while CHAC1 knockdown had reversed effects in K1 cells as indicated by increased cell viability. Taken together, CHAC1 overexpression promoted ferroptosis and enhanced radiation sensitivity in thyroid carcinoma.

Interaction of MRPL9 and GGCT Promotes Cell Proliferation and Migration by Activating the MAPK/ERK Pathway in Papillary Thyroid Cancer.

Thyroid cancer remains the most common endocrine malignancy worldwide, and its incidence has steadily increased over the past four years. Papillary Thyroid Cancer (PTC) is the most common differentiated thyroid cancer, accounting for 80-85% of all thyroid cancers. Mitochondrial proteins (MRPs) are an important part of the structural and functional integrity of the mitochondrial ribosomal complex. It has been reported that MRPL9 is highly expressed in liver cancer and promotes cell proliferation and migration, but it has not been reported in PTC. In the present study we found that MRPL9 was highly expressed in PTC tissues and cell lines, and lentivirus-mediated overexpression of MRPL9 promoted the proliferation and migration ability of PTC cells, whereas knockdown of MRPL9 had the opposite effect. The interaction between MRPL9 and GGCT (γ-glutamylcyclotransferase) was found by immunofluorescence and co-immunoprecipitation experiments (Co-IP). In addition, GGCT is highly expressed in PTC tissues and cell lines, and knockdown of GGCT/MRPL9 in vivo inhibited the growth of subcutaneous xenografts in nude mice and inhibited the formation of lung metastases. Mechanistically, we found that knockdown of GGCT/MRPL9 inhibited the MAPK/ERK signaling pathway. In conclusion, our study found that the interaction of GGCT and MRPL9 modulates the MAPK/ERK pathway, affecting the proliferation and migration of PTC cells. Therefore, GGCT/MRPL9 may serve as a potential biomarker for PTC monitoring and PTC treatment.

DJ-1 inhibits glutathione degradation by downregulating CHAC1 expression in astrocytes.

The PARK7 gene, which encodes DJ-1 protein, is the causative gene of autosomal recessive early-onset Parkinson's disease. DJ-1 has many biological functions, including regulating glutathione (GSH) levels. However, the molecular mechanism by which DJ-1 regulates GSH levels in astrocytes remains unclear. With high throughput sequencing, we discovered that DJ-1 knockout could significantly upregulate the expression of ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1). We demonstrate that DJ-1 can bind with the basic leucine zipper domain of activating transcription factor 3 (ATF3) through bimolecular fluorescence complementation. Besides, DJ-1 inhibits ATF3 binding to the CHAC1 promoter and downregulates the expression of CHAC1 to reduce GSH degradation. Our research suggests that the loss of DJ-1 in astrocytes promotes the degradation of GSH, leading neurons more vulnerable to oxidative damage. It provides a theoretical basis for developing drugs targeting DJ-1 and GSH in the brain.

Administration of Gapmer-type Antisense Oligonucleotides Targeting γ-Glutamylcyclotransferase Suppresses the Growth of A549 Lung Cancer Xenografts.

BACKGROUND/AIM: γ-Glutamyl cyclotransferase (GGCT) is up-regulated in various cancer types, including lung cancer. In this study, we evaluated efficacy of gapmer-type antisense oligonucleotides (ASOs) targeting GGCT in an A549 lung cancer xenograft mouse model and studied their mechanisms of action. MATERIALS AND METHODS: GGCT was inhibited using GGCT-ASOs and cell proliferation was evaluated by dye exclusion test. Western blot analysis was conducted to measure expression of GGCT, p21, p16 and p27, phosphorylation of AMP-activated protein kinase, and caspase activation in A549 cells. Induction of apoptosis and up-regulation of reactive oxygen species were assessed by flow cytometry using annexin V staining and 2',7'-dichlorodihydrofluorescein diacetate dye, respectively. RESULTS: GGCT-ASOs suppressed GGCT expression in A549 cells, inhibited proliferation, and induced apoptosis with activation of caspases. GGCT-ASOs also increased expression of cell-cycle regulating proteins, phospho-AMPK and ROS levels. Systemic administration of GGCT-ASOs to animals bearing A549 lung cancer xenografts showed significant antitumor effects without evident toxicity. CONCLUSION: GGCT-ASOs appear to be promising as novel cancer therapeutic agents.

MiR-205-5p/GGCT Attenuates Growth and Metastasis of Papillary Thyroid Cancer by Regulating CD44.

Papillary thyroid cancer (PTC) remains the most common endocrine malignancy, despite marked achieves in recent decades, and the mechanisms underlying the pathogenesis and progression for PTC are incompletely elucidated. Accumulating evidence show that γ-glutamylcyclotransferase (GGCT), an enzyme participating in glutathione homeostasis and is elevated in multiple types of tumors, represents an attractive therapeutic target. Using bioinformatics, immunohistochemistry, qRT-PCR, and Western blot assays, we found that GGCT expression was upregulated in PTC and correlated with more aggressive clinicopathological characteristics and worse prognosis. GGCT knockdown inhibited the growth and metastasis ability of PTC cells both in vitro and in vivo and reduced the expression of mesenchymal markers (N-cadherin, CD44, MMP2, and MMP9) while increasing epithelial marker (E-cadherin) in PTC cells. We confirmed binding of microRNA-205-5p (miR-205-5p) on the 3'-UTR regions of GGCT by dual-luciferase reporter assay and RNA-RNA pull-down assay. Delivery of miR-205-5p reversed the pro-malignant capacity of GGCT both in vitro and in vivo. Lastly, we found that GGCT interacted with and stabilized CD44 in PTC cells by co-immunoprecipitation and immunohistochemistry assays. Our findings illustrate a novel signaling pathway, miR-205-5p/GGCT/CD44, that involves in the carcinogenesis and progression of PTC. Development of miR-205-mimics or GGCT inhibitors as potential therapeutics for PTC may have remarkable applications.

γ-Glutamylcyclotransferase, a novel regulator of HIF-1α expression, triggers aerobic glycolysis.

Metabolic reprogramming leading to aerobic glycolysis, termed the "Warburg effect," is a critical property of cancer cells. However, the precise mechanisms underlying this phenomenon are not fully understood. A growing body of evidence indicates that γ-glutamylcyclotransferase (GGCT), an enzyme involved in glutathione homeostasis that is highly expressed in many types of cancer, represents a promising therapeutic target. In this study, we identified GGCT as a novel regulator of hypoxia-inducible factor-1α (HIF-1α), a transcription factor that plays a role in hypoxia adaptation promoting aerobic glycolysis. In multiple human cancer cell lines, depletion of GGCT downregulated HIF-1α at the mRNA and protein levels. Conversely, in NIH3T3 mouse fibroblasts, overexpression of GGCT upregulated HIF-1α under normoxia. Moreover, depletion of GGCT downregulated HIF-1α downstream target genes involved in glycolysis, whereas overexpression of GGCT upregulated those genes. Metabolomic analysis revealed that modulation of GGCT expression induced a metabolic switch from the citric acid cycle to glycolysis under normoxia. In addition, we found that GGCT regulates expression of HIF-1α protein via the AMPK-mTORC1-4E-BP1 pathway in PC3 cells. Thus GGCT regulates the expression of HIF-1α in cancer cells, causing a switch to glycolysis.

MITOL/MARCH5 determines the susceptibility of cardiomyocytes to doxorubicin-induced ferroptosis by regulating GSH homeostasis.

MITOL/MARCH5 is an E3 ubiquitin ligase that plays a crucial role in the control of mitochondrial quality and function. However, the significance of MITOL in cardiomyocytes under physiological and pathological conditions remains unclear. First, to determine the significance of MITOL in unstressed hearts, we assessed the cellular changes with the reduction of MITOL expression by siRNA in neonatal rat primary ventricular cardiomyocytes (NRVMs). MITOL knockdown in NRVMs induced cell death via ferroptosis, a newly defined non-apoptotic programmed cell death, even under no stress conditions. This phenomenon was observed only in NRVMs, not in other cell types. MITOL knockdown markedly reduced mitochondria-localized GPX4, a key enzyme associated with ferroptosis, promoting accumulation of lipid peroxides in mitochondria. In contrast, the activation of GPX4 in MITOL knockdown cells suppressed lipid peroxidation and cell death. MITOL knockdown reduced the glutathione/oxidized glutathione (GSH/GSSG) ratio that regulated GPX4 expression. Indeed, the administration of GSH or N-acetylcysteine improved the expression of GPX4 and viability in MITOL-knockdown NRVMs. MITOL-knockdown increased the expression of the glutathione-degrading enzyme, ChaC glutathione-specific γ-glutamylcyclotransferase 1 (Chac1). The knockdown of Chac1 restored the GSH/GSSG ratio, GPX4 expression, and viability in MITOL-knockdown NRVMs. Further, in cultured cardiomyocytes stressed with DOX, both MITOL and GPX4 were reduced, whereas forced-expression of MITOL suppressed DOX-induced ferroptosis by maintaining GPX4 content. Additionally, MITOL knockdown worsened vulnerability to DOX, which was almost completely rescued by treatment with ferrostatin-1, a ferroptosis inhibitor. In vivo, cardiac-specific depletion of MITOL did not produce obvious abnormality, but enhanced susceptibility to DOX toxicity. Finally, administration of ferrostatin-1 suppressed exacerbation of DOX-induced myocardial damage in MITOL-knockout hearts. The present study demonstrates that MITOL determines the cell fate of cardiomyocytes via the ferroptosis process and plays a key role in regulating vulnerability to DOX treatment. (288/300).

miR-877 inhibits the proliferation, migration, and invasion of osteosarcoma cells by targeting gamma-glutamylcyclotransferase.

Gamma-glutamylcyclotransferase (GGCT) can promote the progression of osteosarcoma (OS). MicroRNAs also play significant roles in regulating the progression of OS. This study was designed to investigate whether miR-877 exerts its function in OS by targeting GGCT. The proliferation of OS cells (Saos-2 and U2OS) was detected by MTT and colony formation assays. The migration and invasion of OS cells were detected by transwell assays. The expressions of miRNAs and GGCT were detected by quantitative real-time PCR and Western blot. The luciferase reporter assay was performed to assess whether miR-877 could target GGCT. miR-877 was down-regulated both in OS tissues and OS cell lines (Saos-2 and U2OS). The overexpression of miR-877 inhibited the proliferation, migration, and invasion of OS cell lines, while the knockdown of miR-877 could negate effects. The expression of GGCT was increased in Saos-2 and U2OS cells. miR-877 could target GGCT, and the mRNA level of GGCT in Saos-2 and U2OS cells was decreased by the overexpression of miR-877. miR-877 overexpression inhibited the migration and invasion and suppressed the proliferation of Saos-2 and U2OS cells, and the overexpression of GGCT reversed this effects. The knockdown of miR-877 promoted the migration and invasion and facilitated the proliferation of Saos-2 and U2OS cells, and the silence of GGCT abolished this effects. Our findings suggested that miR-877 could inhibit the proliferation, migration, and invasion of OS cells by targeting GGCT.

Prohibitin 1 is essential to preserve mitochondria and myelin integrity in Schwann cells.

In peripheral nerves, Schwann cells form myelin and provide trophic support to axons. We previously showed that the mitochondrial protein prohibitin 2 can localize to the axon-Schwann-cell interface and is required for developmental myelination. Whether the homologous protein prohibitin 1 has a similar role, and whether prohibitins also play important roles in Schwann cell mitochondria is unknown. Here, we show that deletion of prohibitin 1 in Schwann cells minimally perturbs development, but later triggers a severe demyelinating peripheral neuropathy. Moreover, mitochondria are heavily affected by ablation of prohibitin 1 and demyelination occurs preferentially in cells with apparent mitochondrial loss. Furthermore, in response to mitochondrial damage, Schwann cells trigger the integrated stress response, but, contrary to what was previously suggested, this response is not detrimental in this context. These results identify a role for prohibitin 1 in myelin integrity and advance our understanding about the Schwann cell response to mitochondrial damage.

Transcriptomic analysis of the effect of (E)-3-(3,5-dimethoxyphenyl)-1-(2-methoxyphenyl) prop-2-en-1-one (DPP23) on reactive oxygen species generation in MIA PaCa-2 pancreatic cancer cells.

BACKGROUND: Reactive oxygen species (ROS) generation specifically in cancer cells may be a promising strategy for their selective killing. The synthetic chalcone derivative (E)-3-(3,5-dimethoxyphenyl)-1-(2-methoxyphenyl)prop-2-en-1-one (DPP23) exerts antitumor activity through ROS-mediated apoptosis in cancer cells but not in healthy cells. However, the mechanism underlying ROS generation by DPP23 remains unknown. OBJECTIVE: The current study aims to identify possible DPP23 target genes responsible for ROS generation through the mining of microarray data stored in NCBI's Gene Expression Omnibus (GEO). METHODS: A comprehensive expression profile of genes modulated by DPP23 was examined by gene ontology analysis. DPP23-modulated genes in Mia-PaCa2 pancreatic cells were validated by reverse transcription-PCR. RESULTS: Multiple genes were up and downregulated by DPP23 treatment in MiaPaCa2 pancreatic cancer cells. Genes with absolute fold-change (FC) of > 2 were selected as the cut-off criteria and grouped into 10 clusters to analyze expression patterns systematically. We observed that genes with increased expression at 6 h were significantly affected by ROS increase, unfolded protein response, and cell death. Expression of 13 genes involved in glutathione metabolism, including CHAC1, GCLC, G6PD, GSTO2, GSTA5, GSTM2, GSR, GPX3/6/8, GGT1, PGD, ATF4, and NAT8B, are modulated by DPP23. Of these, CHAC1 was most highly upregulated upon DPP23 treatment. CONCLUSION: DPP23 alters global gene expression associated with multiple cellular responses, including oxidative stress and apoptosis. We found that DPP23 may induce GSH depletion through modulation of gene expression, which is especially involved in glutathione metabolism. Of these, CHAC1 emerged as the most prominent candidate for DPP23 as it was the most responsive to DPP23 treatment.

γ-Glutamyl cyclotransferase contributes to endometrial carcinoma malignant progression and upregulation of PD-L1 expression during activation of epithelial-mesenchymal transition.

Recent increases in the incidence of endometrial carcinoma represent a significant risk to women's health. We found that γ-glutamyl cyclotransferase (GGCT) was significantly up-regulated in endometrial carcinoma tissues and cells, which suggested that it may be a potential target for treatment of endometrial carcinoma. Furthermore, the impact of GGCT on proliferation, migration, and invasion of endometrial carcinoma has been demonstrated in vitro and in vivo using GGCT silencing and overexpression techniques. In addition, the epithelial-mesenchymal transition (EMT) was significantly inhibited in response to GGCT knockdown, which indicated that GGCT may contribute endometrial carcinoma malignancy during activation of the EMT. We also found that GGCT regulated PD-L1 expression during EMT activation. Furthermore, co-culture of endometrial carcinoma cells with CD8+ T lymphocytes showed that downregulation of PD-L1 expression following GGCT knockdown contributed to the killing activity of CD8+ T lymphocytes on endometrial carcinoma cells. In conclusion, our study showed that GGCT contributed to malignant progression and upregulation of PD-L1 expression of endometrial carcinoma, and may be a potential target for treatment of endometrial carcinoma.

Active mixture of serum-circulating small molecules selectively inhibits proliferation and triggers apoptosis in cancer cells via induction of ER stress.

Genetic and epigenetic regulation as well as immune surveillance are known defense mechanisms to protect organisms from developing cancer. Based on experimental evidence, we proposed that small metabolically active molecules accumulating in cancer cells may play a role in an alternative antitumor surveillance system. Previously, we reported that treatment with a mixture of experimentally selected small molecules, usually found in the serum (defined 'active mixture', AM), selectively induces apoptosis in cancer cells and significantly inhibits tumor formation in vivo. In this study, we show that the AM elicits gene expression changes characteristic of endoplasmic reticulum (ER) stress in HeLa, MCF-7, PC-3 and Caco-2 cancer cells, but not in primary human renal epithelial cells. The activation of the ER stress pathway was confirmed by the upregulation of ATF3, ATF4, CHAC1, DDIT3 and GDF15 proteins. Mechanistically, our investigation revealed that eIF2α, PERK and IRE1α are phosphorylated upon treatment with the AM, linking the induction of ER stress to the antiproliferative and proapoptotic effects of the AM previously demonstrated. Inhibition of ER stress in combination with BBC3 and PMAIP1 knockdown completely abrogated the effect of the AM. Moreover, we also demonstrated that the AM induces mIR-3189-3p, which in turn enhances the expression of ATF3 and DDIT3, thus representing a possible new feedback mechanism in the regulation of ATF3 and DDIT3 during ER stress. Our results highlight small molecules as attractive anticancer agents and warrant further evaluation of the AM in cancer therapy, either alone or in combination with other ER stress inducing agents.

Structural and Functional Analyses of Human ChaC2 in Glutathione Metabolism.

Glutathione (GSH) degradation plays an essential role in GSH homeostasis, which regulates cell survival, especially in cancer cells. Among human GSH degradation enzymes, the ChaC2 enzyme acts on GSH to form 5-l-oxoproline and Cys-Gly specifically in the cytosol. Here, we report the crystal structures of ChaC2 in two different conformations and compare the structural features with other known γ-glutamylcyclotransferase enzymes. The unique flexible loop of ChaC2 seems to function as a gate to achieve specificity for GSH binding and regulate the constant GSH degradation rate. Structural and biochemical analyses of ChaC2 revealed that Glu74 and Glu83 play crucial roles in directing the conformation of the enzyme and in modulating the enzyme activity. Based on a docking study of GSH to ChaC2 and binding assays, we propose a substrate-binding mode and catalytic mechanism. We also found that overexpression of ChaC2, but not mutants that inhibit activity of ChaC2, significantly promoted breast cancer cell proliferation, suggesting that the GSH degradation by ChaC2 affects the growth of breast cancer cells. Our structural and functional analyses of ChaC2 will contribute to the development of inhibitors for the ChaC family, which could effectively regulate the progression of GSH degradation-related cancers.

Blockade of γ-Glutamylcyclotransferase Enhances Docetaxel Growth Inhibition of Prostate Cancer Cells.

BACKGROUND/AIM: γ-Glutamylcyclotransferase (GGCT) is highly expressed in many forms of cancer, and is a promising therapeutic target. The present study investigated whether inhibition of GGCT enhanced the antiproliferative effects of the drug docetaxel in prostate cancer cells. MATERIALS AND METHODS: Immunohistochemistry and western blot analysis were conducted to measure GGCT expression in prostate cancer tissue samples and cell lines. GGCT was inhibited using RNAi and a novel enzymatic inhibitor, pro-GA, and cell proliferation was evaluated with single and combination treatments of GGCT inhibitors and docetaxel. RESULTS: GGCT was highly expressed in cultured prostate cancer cells and patient samples. GGCT inhibition alone inhibited prostate cancer cell line proliferation and induced cellular senescence. GGCT inhibition in combination with apoptosis-inducing docetaxel had more potent antiproliferative effects than either drug used alone. CONCLUSION: GGCT inhibition may potentiate anticancer drug efficacy.

Depletion of gamma-glutamylcyclotransferase inhibits cancer cell growth by activating the AMPK-FOXO3a-p21 axis.

Inhibition of gamma-glutamylcyclotransferase (GGCT), which is highly expressed in various cancer tissues, exerts anticancer effects both in vitro and in vivo. Previous studies have shown that depletion of GGCT blocks the growth of MCF7 breast cancer cells via upregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21); in addition, induction of autophagy plays a role in the upregulation of p21 upon GGCT knockdown. However, the mechanisms underlying induction of p21 in cancer cells are not fully understood. Here, we show that GGCT knockdown in PC3 human prostate cancer and A172 glioblastoma cells upregulates the mRNA and nuclear protein levels of Forkhead box O transcription factor 3a (FOXO3a), a transcriptional factor involved in tumor suppression. Simultaneous knockdown of FOXO3a and GGCT in PC3 and A172 cells attenuated upregulation of p21, followed by growth inhibition and cell death. Furthermore, simultaneous knockdown of GGCT and AMP-activated protein kinase (AMPK) α, a metabolic stress sensor, in PC3 and A172 cells led to marked attenuation of cellular responses induced by GGCT knockdown, including an increase in FOXO3a phosphorylation at Ser413, upregulation of p21, growth inhibition, and cell death. These results indicate that the AMPK-FOXO3a-p21 axis plays an important role in inhibition of cancer cell growth by depletion of GGCT.