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.

E3 ubiquitin ligase RNF148 functions as an oncogene in colorectal cancer by ubiquitination-mediated degradation of CHAC2.

We previously reported that RNF148 was involved in the ubiquitination-mediated degradation of CHAC2. However, its molecular mechanism was not determined. In this study, we investigated the role and mechanism of RNF148 in the progression of colorectal cancer (CRC), especially in the process of ubiquitination-mediated degradation of CHAC2. Our results revealed that RNF148 was upregulated in most CRC tissues, and its expression significantly correlated with the 3-year overall survival rate and most clinicopathological parameters of CRC patients. Furthermore, RNF148 served as an independent prognostic biomarker of CRC and promoted CRC cell proliferation and migration while inhibiting cell apoptosis and sensitivity to 5-FU. Mechanistically, RNF148 used its protease-associated domain to bind to the CHAC domain of CHAC2 and target it for degradation. In addition, we identified two phosphorylation and three ubiquitination residues of CHAC2 and identified Y118 and K102 as the critical phosphorylation and ubiquitination residues, respectively. We also identified CHAC2's and RNF148's interacting proteins and discovered their potential interaction network. In conclusion, our current study unveiled the role of RNF148 in CRC and the mechanism of RNF148 in the ubiquitination-mediated degradation of CHAC2, which shed light on providing potential prognostic biomarkers and molecular targets for CRC patients.

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.

Development of an activity-based chemiluminogenic probe for γ-glutamylcyclotransferase.

While γ-glutamylcyclotransferase (GGCT) has been implicated in cancer-cell proliferation, the role of GGCT enzymatic activity in the regulation of cancer-cell growth remains unclear. Toward further understanding of GGCT in vivo, here we report a novel cell-permeable chemiluminogenic probe "MAM-LISA-103" that detects intracellular GGCT activity and apply it to in vivo imaging. We first developed a chemiluminogenic probe LISA-103, which simply and sensitively detects the enzymatic activity of recombinant GGCT through chemiluminescence. We then designed the cell-permeable GGCT probe MAM-LISA-103 and applied it to several biological experiments. MAM-LISA-103 successfully detected the intracellular GGCT activity in GGCT-overexpressing NIH-3T3 cells. Moreover, MAM-LISA-103 demonstrated tumor-imaging ability when administered to a xenograft model using immunocompromised mice inoculated with MCF7 cells.

Identification of c-Met as a novel target of γ-glutamylcyclotransferase.

γ-Glutamylcyclotransferase (GGCT) is highly expressed in multiple types of cancer tissues and its knockdown suppresses the growth of cancer cells in vitro and in vivo. Although GGCT is a promising target for cancer therapy, the mechanisms underlying the antitumor effects remain unclear. The knockdown of GGCT inhibited the MEK-ERK pathway, and activated the tumor suppressor retinoblastoma gene (RB) at the protein level in cancer cell lines. c-Met was down-regulated by the knockdown of GGCT in cancer cells and its overexpression attenuated the dephosphorylation of RB and cell cycle arrest induced by the knockdown of GGCT in lung cancer A549 cells. STAT3 is a transcription factor that induces c-Met expression. STAT3 phosphorylation and its nuclear expression level were decreased in GGCT-depleted A549 and prostate cancer PC3 cells. The simultaneous knockdown of AMPK and GGCT restored the down-regulated expression of c-Met, and attenuated the dephosphorylation of STAT3 and MEK-ERK-RB induced by the knockdown of GGCT in PC3 cells. An intraperitoneal injection of a GGCT inhibitor decreased c-Met protein expression in a mouse xenograft model of PC3 cells. These results suggest that the knockdown of GGCT activates the RB protein by inhibiting the STAT3-c-Met-MEK-ERK pathway via AMPK activation.

CHAC1 inactivation is effective to preserve muscle glutathione but is insufficient to protect against muscle wasting in cachexia.

Muscle wasting is one of the main characteristics of cachexia associated with cancer and other chronic diseases and is often exacerbated by antineoplastic agents. Increased oxidative stress is associated with muscle wasting, along with depletion of glutathione, the most abundant endogenous antioxidant. Therefore, boosting endogenous glutathione has been proposed as a therapeutic strategy to prevent muscle wasting. Here, we tested this hypothesis by inactivating CHAC1, an intracellular glutathione degradation enzyme. We found CHAC1 expression is increased under multiple muscle wasting conditions in animal models, including fasting, cancer cachexia, and chemotherapy. The elevation of muscle Chac1 expression is associated with reduced glutathione level. CHAC1 inhibition via CRSPR/Cas9 mediated knock-in of an enzyme inactivating mutation demonstrates a novel strategy to preserve muscle glutathione levels under wasting conditions but fails to prevent muscle wasting in mice. These results suggest that preserving intracellular glutathione level alone may not be sufficient to prevent cancer or chemotherapy induced muscle wasting.

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.

High levels of unfolded protein response component CHAC1 associates with cancer progression signatures in malignant breast cancer tissues

Purpose The aberrant mRNA expression of a UPR component Cation transport regulator homolog 1 (CHAC1) has been reported to be associated with poor survival in breast and ovarian cancer patients, however, the expression of CHAC1 at protein levels in malignant breast tissues is underreported. The following study aimed at analyzing CHAC1 protein expression in malignant breast cancer tissues. Method Evaluation of CHAC1 expression in invasive ductal carcinomas (IDCs) with known ER, PR, and HER2 status was carried out using immunohistochemistry (IHC) with CHAC1 specific antibody. The Human breast cancer tissue microarray (TMA, cat# BR1503f, US Biomax, Inc., Rockville, MD) was used to determine CHAC1 expression. The analysis of CHAC1 IHC was done to determine its expression in terms of molecular subtypes of breast cancer, lymph node status, and proliferation index using Qu-Path software. Survival analysis was studied with a Kaplan–Meier plotter. Results Immunohistochemical analysis of CHAC1 in breast cancer tissues showed significant up-regulation of CHAC1 as compared to the adjacent normal and benign tissues. Interestingly, CHAC1 immunostaining revealed high expression in tumor tissues with high proliferation and positive lymph node metastasis suggesting that CHAC1 might have an important role to play in breast cancer progression. Furthermore, high CHAC1 expression is associated with poor overall survival (OS) in large breast cancer patient cohorts. Conclusion As a higher expression of CHAC1 was observed in tissue cores with high Ki67 index and positive lymph node metastasis it may be concluded that enhanced CHAC1 expression correlates with proliferation and metastasis. The further analysis of breast cancer patients’ survival data through KM plot indicated that high CHAC1 expression is associated with a bad prognosis hinting that CHAC1 may have a possible prognostic significance in breast cancer (AU)

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.

The γ-Glutamylcyclotransferase Inhibitor Pro-GA Induces an Antiproliferative Effect Through the Generation of Mitochondrial Reactive Oxygen Species.

BACKGROUND/AIM: γ-Glutamylcyclotransferase (GGCT) is up-regulated in a broad range of cancers, including breast cancer, and GGCT inhibition has been shown to be a promising strategy for therapy. Herein, we evaluated the efficacy and mechanism of action of pro-GA, a GGCT enzymatic inhibitor, in MCF7 breast cancer cells. MATERIALS AND METHODS: Proliferation was evaluated by WST-8 and trypan blue dye exclusion assays. Western blot analysis was conducted to examine the expression of cyclin-dependent kinase inhibitors (CDKI), including p21, p27, and p16. Induction of senescence was assessed by senescence-associated ß-galactosidase staining. Generation of mitochondrial superoxide reactive oxygen species (ROS) was assessed using flow cytometry. The effect of N-acetylcysteine (NAC) on pro-GA dependent inhibition of proliferation, ROS generation, and senescence was also studied. The efficacy of systemic administration of pro-GA was evaluated in a MCF7 xenograft mouse model. RESULTS: Treatment with pro-GA inhibited proliferation of MCF7 cells, increased CDKI expression and mitochondrial ROS, and induced cellular senescence. We found that cotreatment with NAC restored proliferation in pro-GA treated cells. NAC similarly suppressed CDKI expression, mitochondrial ROS generation, and senescence induced by pro-GA. Furthermore, the systemic administration of pro-GA in an MCF7 xenograft model had significant antitumor effects without toxicity. CONCLUSION: Pro-GA may be a promising therapeutic agent for the treatment of breast cancer.

High levels of unfolded protein response component CHAC1 associates with cancer progression signatures in malignant breast cancer tissues.

PURPOSE: The aberrant mRNA expression of a UPR component Cation transport regulator homolog 1 (CHAC1) has been reported to be associated with poor survival in breast and ovarian cancer patients, however, the expression of CHAC1 at protein levels in malignant breast tissues is underreported. The following study aimed at analyzing CHAC1 protein expression in malignant breast cancer tissues. METHODS: Evaluation of CHAC1 expression in invasive ductal carcinomas (IDCs) with known ER, PR, and HER2 status was carried out using immunohistochemistry (IHC) with CHAC1 specific antibody. The Human breast cancer tissue microarray (TMA, cat# BR1503f, US Biomax, Inc., Rockville, MD) was used to determine CHAC1 expression. The analysis of CHAC1 IHC was done to determine its expression in terms of molecular subtypes of breast cancer, lymph node status, and proliferation index using Qu-Path software. Survival analysis was studied with a Kaplan-Meier plotter. RESULTS: Immunohistochemical analysis of CHAC1 in breast cancer tissues showed significant up-regulation of CHAC1 as compared to the adjacent normal and benign tissues. Interestingly, CHAC1 immunostaining revealed high expression in tumor tissues with high proliferation and positive lymph node metastasis suggesting that CHAC1 might have an important role to play in breast cancer progression. Furthermore, high CHAC1 expression is associated with poor overall survival (OS) in large breast cancer patient cohorts. CONCLUSION: As a higher expression of CHAC1 was observed in tissue cores with high Ki67 index and positive lymph node metastasis it may be concluded that enhanced CHAC1 expression correlates with proliferation and metastasis. The further analysis of breast cancer patients' survival data through KM plot indicated that high CHAC1 expression is associated with a bad prognosis hinting that CHAC1 may have a possible prognostic significance in breast cancer.

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.

Glaucocalyxin A impairs tumor growth via amplification of the ATF4/CHOP/CHAC1 cascade in human oral squamous cell carcinoma.

ETHNOPHARMACOLOGICAL RELEVANCE: The natural extract glaucocalyxin A (GLA), purified from the aboveground sections of the Chinese traditional medicinal herb Rabdosia japonica (Burm. f.) Hara var. glaucocalyx (Maxim.) Hara, has various pharmacological benefits, such as anti-bacterial, anti-coagulative, anti-neoplastic, and anti-inflammatory activities. Although GLA has shown anti-tumor activity against various cancers, the therapeutic potential and biological mechanisms of GLA remain to be further explored in oral squamous cell carcinoma (OSCC). AIM OF THE STUDY: This study aimed to elucidate the therapeutic potential and regulatory mechanisms of GLA in OSCC. MATERIALS AND METHODS: The cell proliferation and apoptosis effects of GLA were analyzed by CCK-8, clone formation, Annexin V/PI staining, and apoptotic protein expression in vitro. An OSCC xenograft model was applied to confirm the anti-neoplastic effect in vivo. Furthermore, the changes of reactive oxygen species (ROS) were determined by DCFH-DA probe and GSH/GSSG assay, and inhibited by the pan-caspase inhibitor Z-VAD(OMe)-FMK and the ROS scavenger N-acetylcysteine (NAC). The modulation of GLA on mitochondria and ER-dependent apoptosis pathways was analyzed by JC-1 probe, quantitative real-time PCR, and Western blot. Finally, public databases, clinical samples, and transfection cells were analyzed to explore the importance of GLA's indirect targeting molecule CHAC1 in OSCC. RESULTS: GLA significantly inhibited cell proliferation and induced apoptosis in vitro and in vivo. GLA perturbed the redox homeostasis, and cell apoptosis was totally rescued by Z-VAD(OMe)-FMK and NAC. Furthermore, GLA activated the mitochondrial apoptosis pathway. Simultaneously, the overexpression and knockdown of CHAC1 dramatically affected GLA-mediated apoptosis. The endoplasmic reticulum stress-associated ATF4/CHOP signal was identified to participate in GLA-upregulated CHAC1 expression. Finally, we found that CHAC1 expression was lower in OSCC compared with normal tissues and positively correlated with 4-Hydroxynonenal (4-HNE) level. High CHAC1 expression also indicated better overall survival. Moreover, CHAC1 selectively regulated the viability of oral cancer cells. CONCLUSION: GLA is a promising therapeutic agent that activates the ROS-mediated ATF4/CHOP/CHAC1 axis in OSCC patients.

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.

Fluorizoline Blocks the Interaction between Prohibitin-2 and γ-Glutamylcyclotransferase and Induces p21Waf1/Cip1 Expression in MCF7 Breast Cancer Cells.

Prohibitin-2 (PHB2) is a scaffold protein that has pleiotropic functions, which include interacting with γ-glutamylcyclotransferase (GGCT) in the cytoplasm and repressing the transcriptional activities of the p21Waf1/Cip (p21) gene in the nucleus. The cytotoxic drug fluorizoline binds to PHB1/2 and exerts antiproliferative actions on cancer cells. However, the precise mechanism underlying the antiproliferative effects of fluorizoline is not fully elucidated. In the present study, we first show that fluorizoline induces p21 expression in several human cancer cell lines, including MCF7 breast cancer cells. Treatment of MCF7 cells with fluorizoline suppressed proliferation and prevented cells from entering into the DNA synthesis phase. Knockdown of p21 rescued the suppressed proliferation, indicating that fluorizoline inhibited MCF7 cell growth via the induction of p21. Overexpression of PHB2 in MCF7 cells prevented the induction of p21 expression by fluorizoline and restored the antiproliferative effects and blockade of cell cycle progression. Moreover, treatment of MCF7 cells with fluorizoline inhibited the interaction between endogenous PHB2 and GGCT proteins and reduced the level of nuclear localization of PHB2 proteins. These results indicate that targeting PHB2 with fluorizoline induces the expression of p21 and consequently blocks proliferation of cancer cells. SIGNIFICANCE STATEMENT: This study shows that fluorizoline may be a promising novel anticancer drug candidate that induces p21 expression and blocks cell-cycle progression in human cancer cell lines. In addition, we show that fluorizoline inhibits the interaction between PHB2 and GGCT and reduces the nuclear localization of PHB2 proteins.