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Li M, Wang X, Chen X, Hong J, Du Y, Song D. GK921, a transglutaminase inhibitor, strengthens the antitumor effect of cisplatin on pancreatic cancer cells by inhibiting epithelial-to-mesenchymal transition. Biochim Biophys Acta Mol Basis Dis 2024; 1870:166925. [PMID: 38084873 DOI: 10.1016/j.bbadis.2023.166925] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2023] [Revised: 10/02/2023] [Accepted: 10/20/2023] [Indexed: 12/30/2023]
Abstract
Pancreatic adenocarcinoma (PAAD), a common digestive malignant tumor, presents high mortality rates and limited treatment methods. Currently, chemotherapy remains the main therapy method for patients with PAAD. As a classical chemotherapy drug, cisplatin (DDP) is limited by dose-related toxicity in patients with PAAD. In this study, we demonstrated that TGM2 may be a treatment and prognosis marker in pancreatic cancer patients. Co-treatment of low dose of DDP and GK921, a transglutaminase (TGM2) inhibitor, is capable of synergistically inhibiting the PAAD cell viability and proliferation in vitro and in vivo. Based on in vitro study, GK921 inhibited the epithelial-to-mesenchymal transition (EMT) induced by TGM2 as well as aggravated cell cycle arrest and apoptosis resulted from DDP, making pancreatic cancer cells more sensible to DDP. Our results showed that GK921 increased the protein levels regarding E-cadherin as well as decreased the protein level regarding Snail2, N-cadherin, which indicated that GK921 inhibited EMT in pancreatic cancer cells. Snail2 overexpression inhibited GK921/DDP-induced cell apoptosis, as well as mitigated the GK921/DDP-caused cell death and the EMT inhibition. In vivo studies also found GK921/DDP combination can further inhibit the growth of PAAD without significantly side effects. To sum up, we showed that GK921 increased PAAD cells sensitivity to DDP via inhibiting EMT. As revealed, DDP/GK921 co-treatment could promisingly serve for treating PAAD patients.
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Affiliation(s)
- Mengxin Li
- Department of Breast Surgery, General Surgery Center, First Hospital of Jilin University, Changchun, China
| | - Xuanzhong Wang
- Department of Radiation Oncology, First Hospital of Jilin University, Changchun, China
| | - Xuyang Chen
- School of Basic Medicine and Life Sciences, Hainan Medical University, Haikou, China
| | - Jinghui Hong
- Department of Breast Surgery, General Surgery Center, First Hospital of Jilin University, Changchun, China
| | - Ye Du
- Department of Breast Surgery, General Surgery Center, First Hospital of Jilin University, Changchun, China
| | - Dong Song
- Department of Breast Surgery, General Surgery Center, First Hospital of Jilin University, Changchun, China.
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2
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Spotlight on the transglutaminase 2 gene: a focus on genomic and transcriptional aspects. Biochem J 2018; 475:1643-1667. [PMID: 29764956 DOI: 10.1042/bcj20170601] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2017] [Revised: 04/10/2018] [Accepted: 04/12/2018] [Indexed: 01/16/2023]
Abstract
The type 2 isoenzyme is the most widely expressed transglutaminase in mammals displaying several intra- and extracellular activities depending on its location (protein modification, modulation of gene expression, membrane signalling and stabilization of cellular interactions with the extracellular matrix) in relation to cell death, survival and differentiation. In contrast with the appreciable knowledge about the regulation of the enzymatic activities, much less is known concerning its inducible expression, which is altered in inflammatory and neoplastic diseases. In this context, we first summarize the gene's basic features including single-nucleotide polymorphism characterization, epigenetic DNA methylation and identification of regulatory regions and of transcription factor-binding sites at the gene promoter, which could concur to direct gene expression. Further aspects related to alternative splicing events and to ncRNAs (microRNAs and lncRNAs) are involved in the modulation of its expression. Notably, this important gene displays transcriptional variants relevant for the protein's function with the occurrence of at least seven transcripts which support the synthesis of five isoforms with modified catalytic activities. The different expression of the TG2 (type 2 transglutaminase) variants might be useful for dictating the multiple biological features of the protein and their alterations in pathology, as well as from a therapeutic perspective.
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Minotti L, Baldassari F, Galasso M, Volinia S, Bergamini CM, Bianchi N. A long non-coding RNA inside the type 2 transglutaminase gene tightly correlates with the expression of its transcriptional variants. Amino Acids 2018; 50:421-438. [PMID: 29313085 DOI: 10.1007/s00726-017-2528-9] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2017] [Accepted: 12/10/2017] [Indexed: 12/13/2022]
Abstract
The long non-coding RNAs (lncRNAs) are matter of intense investigation as potential regulators of gene expression. In the case of the transglutaminase 2 gene (TGM2) the databases of genome sequence indicate location of a lncRNA (LOC107987281) within the first intron. This lncRNA is 1000 bp long, arises from 2 exons and starts few nucleotides 3' of the first splicing site of translated TGM2. We have analysed correlations between expression of LOC107987281 lncRNA and TGM2 mRNA by real-time PCR in K562 cell line untreated or treated with the anticancer drugs TPA (12-O-tetradecanoylphorbol-13-acetate), Docetaxel and Doxorubicin. In the treated cells the lncRNA increase follows the trend of TGM2 transcript. To validate this finding we used HumanExon1_0ST Affymetrix; chip data were background-adjusted, quantile-normalized and summarized using robust multi-array average analysis implemented in the R package. The probesets recognize sequences inside each exon, near intronic splicing sites and others located in the untranslated regions of TGM2 gene. The analysis of total RNA samples in GEO datasets from K562, HL-60, THP-1 and U937 cell lines, untreated or treated with TPA in replicated experiments confirmed our earlier results. These demonstrate correlation between LOC107987281 and TGM2 mRNA in the cell lines (K562, HL60 and THP-1) where increased levels of TGM2 mRNA are produced. Additional array study on 358 samples of several normal and paired tumor tissues leads to the same conclusions, indicating a correlation between full-length TGM2 mRNA and LOC107987281 lncRNA in relation to the development of several tumors.
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Affiliation(s)
- Linda Minotti
- Section of Anatomy and Histology, Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy
| | - Federica Baldassari
- Section of Anatomy and Histology, Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy
| | - Marco Galasso
- Section of Anatomy and Histology, Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy
| | - Stefano Volinia
- Section of Anatomy and Histology, Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy
| | - Carlo M Bergamini
- Section of Biochemistry, Molecular Biology and Medical Genetics, Department of Biomedical Sciences and Specialist Surgery, University of Ferrara, Via Luigi Borsari 46, 44121, Ferrara, Italy
| | - Nicoletta Bianchi
- Section of Biochemistry, Molecular Biology and Medical Genetics, Department of Biomedical Sciences and Specialist Surgery, University of Ferrara, Via Luigi Borsari 46, 44121, Ferrara, Italy.
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4
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Thangaraju K, Király R, Demény MA, András Mótyán J, Fuxreiter M, Fésüs L. Genomic variants reveal differential evolutionary constraints on human transglutaminases and point towards unrecognized significance of transglutaminase 2. PLoS One 2017; 12:e0172189. [PMID: 28248968 PMCID: PMC5332030 DOI: 10.1371/journal.pone.0172189] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2016] [Accepted: 02/01/2017] [Indexed: 01/16/2023] Open
Abstract
Transglutaminases (TGMs) catalyze Ca2+-dependent transamidation of proteins with specified roles in blood clotting (F13a) and in cornification (TGM1, TGM3). The ubiquitous TGM2 has well described enzymatic and non-enzymatic functions but in-spite of numerous studies its physiological function in humans has not been defined. We compared data on non-synonymous single nucleotide variations (nsSNVs) and loss-of-function variants on TGM1-7 and F13a from the Exome aggregation consortium dataset, and used computational and biochemical analysis to reveal the roles of damaging nsSNVs of TGM2. TGM2 and F13a display rarer damaging nsSNV sites than other TGMs and sequence of TGM2, F13a and TGM1 are evolutionary constrained. TGM2 nsSNVs are predicted to destabilize protein structure, influence Ca2+ and GTP regulation, and non-enzymatic interactions, but none coincide with conserved functional sites. We have experimentally characterized six TGM2 allelic variants detected so far in homozygous form, out of which only one, p.Arg222Gln, has decreased activities. Published exome sequencing data from various populations have not uncovered individuals with homozygous loss-of-function variants for TGM2, TGM3 and TGM7. Thus it can be concluded that human transglutaminases differ in harboring damaging variants and TGM2 is under purifying selection suggesting that it may have so far not revealed physiological functions.
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Affiliation(s)
- Kiruphagaran Thangaraju
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
| | - Róbert Király
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
| | - Máté A. Demény
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
| | - János András Mótyán
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
| | - Mónika Fuxreiter
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
- MTA-DE Momentum Laboratory of Protein Dynamics, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
| | - László Fésüs
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
- MTA-DE Stem cell, Apoptosis and Genomics Research Group of Hungarian Academy of Sciences, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
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Tatsukawa H, Furutani Y, Hitomi K, Kojima S. Transglutaminase 2 has opposing roles in the regulation of cellular functions as well as cell growth and death. Cell Death Dis 2016; 7:e2244. [PMID: 27253408 PMCID: PMC5143380 DOI: 10.1038/cddis.2016.150] [Citation(s) in RCA: 89] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2016] [Revised: 04/28/2016] [Accepted: 04/28/2016] [Indexed: 01/27/2023]
Abstract
Transglutaminase 2 (TG2) is primarily known as the most ubiquitously expressed member of the transglutaminase family with Ca2+-dependent protein crosslinking activity; however, this enzyme exhibits multiple additional functions through GTPase, cell adhesion, protein disulfide isomerase, kinase, and scaffold activities and is associated with cell growth, differentiation, and apoptosis. TG2 is found in the extracellular matrix, plasma membrane, cytosol, mitochondria, recycling endosomes, and nucleus, and its subcellular localization is an important determinant of its function. Depending upon the cell type and stimuli, TG2 changes its subcellular localization and biological activities, playing both anti- and pro-apoptotic roles. Increasing evidence indicates that the GTP-bound form of the enzyme (in its closed form) protects cells from apoptosis but that the transamidation activity of TG2 (in its open form) participates in both facilitating and inhibiting apoptosis. A difficulty in the study and understanding of this enigmatic protein is that opposing effects have been reported regarding its roles in the same physiological and/or pathological systems. These include neuroprotective or neurodegenerative effects, hepatic cell growth-promoting or hepatic cell death-inducing effects, exacerbating or having no effect on liver fibrosis, and anti- and pro-apoptotic effects on cancer cells. The reasons for these discrepancies have been ascribed to TG2's multifunctional activities, genetic variants, conformational changes induced by the immediate environment, and differences in the genetic background of the mice used in each of the experiments. In this article, we first report that TG2 has opposing roles like the protagonist in the novel Dr. Jekyll and Mr. Hyde, followed by a summary of the controversies reported, and finally discuss the possible reasons for these discrepancies.
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Affiliation(s)
- H Tatsukawa
- Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Nagoya 464-8601, Japan
| | - Y Furutani
- Micro-Signaling Regulation Technology Unit, RIKEN Center for Life Science Technologies, 2-1 Hirosawa, Saitama 351-0198, Japan
| | - K Hitomi
- Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Nagoya 464-8601, Japan
| | - S Kojima
- Micro-Signaling Regulation Technology Unit, RIKEN Center for Life Science Technologies, 2-1 Hirosawa, Saitama 351-0198, Japan
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6
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Király R, Thangaraju K, Nagy Z, Collighan R, Nemes Z, Griffin M, Fésüs L. Isopeptidase activity of human transglutaminase 2: disconnection from transamidation and characterization by kinetic parameters. Amino Acids 2015; 48:31-40. [PMID: 26250429 DOI: 10.1007/s00726-015-2063-5] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2015] [Accepted: 07/27/2015] [Indexed: 12/24/2022]
Abstract
Transglutaminase 2 (TG2) is a multifunctional protein with diverse catalytic activities and biological roles. Its best studied function is the Ca(2+)-dependent transamidase activity leading to formation of γ-glutamyl-ε-lysine isopeptide crosslinks between proteins and γ-glutamyl-amine derivatives. TG2 has a poorly studied isopeptidase activity cleaving these bonds. We have developed and characterised TG2 mutants which are significantly deficient in transamidase activity while have normal or increased isopeptidase activity (W332F) and vice versa (W278F). The W332F mutation led to significant changes of both the K m and the V max kinetic parameters of the isopeptidase reaction of TG2 while its calcium and GTP sensitivity was similar to the wild-type enzyme. The W278F mutation resulted in six times elevated amine incorporating transamidase activity demonstrating the regulatory significance of W278 and W332 in TG2 and that mutations can change opposed activities located at the same active site. The further application of our results in cellular systems may help to understand TG2-driven physiological and pathological processes better and lead to novel therapeutic approaches where an increased amount of crosslinked proteins correlates with the manifestation of degenerative disorders.
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Affiliation(s)
- Róbert Király
- Department of Biochemistry and Molecular Biology, University of Debrecen, Egyetem tér 1., Debrecen, 4012, Hungary
| | - Kiruphagaran Thangaraju
- Department of Biochemistry and Molecular Biology, University of Debrecen, Egyetem tér 1., Debrecen, 4012, Hungary
| | - Zsófia Nagy
- Department of Biochemistry and Molecular Biology, University of Debrecen, Egyetem tér 1., Debrecen, 4012, Hungary
| | - Russell Collighan
- School of Life and Health Sciences, Aston University, Birmingham, UK
| | - Zoltán Nemes
- Department of Biochemistry and Molecular Biology, University of Debrecen, Egyetem tér 1., Debrecen, 4012, Hungary
| | - Martin Griffin
- School of Life and Health Sciences, Aston University, Birmingham, UK
| | - László Fésüs
- Department of Biochemistry and Molecular Biology, University of Debrecen, Egyetem tér 1., Debrecen, 4012, Hungary. .,MTA-DE Stem Cell, Apoptosis and Genomics Research Group of Hungarian Academy of Sciences, Faculty of Medicine, University of Debrecen, Egyetem tér 1., Debrecen, 4012, Hungary.
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7
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Zhu L, Qu XH, Sun YL, Qian YM, Zhao XH. Novel method for extracting exosomes of hepatocellular carcinoma cells. World J Gastroenterol 2014; 20:6651-6657. [PMID: 24914390 PMCID: PMC4047354 DOI: 10.3748/wjg.v20.i21.6651] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/31/2013] [Revised: 02/08/2014] [Accepted: 03/05/2014] [Indexed: 02/06/2023] Open
Abstract
AIM: To develop a novel method for the rapid and efficient extraction of exosomes secreted by tumor cells.
METHODS: Unlike the traditional extraction method, the supernatants of cell cultures were concentrated, and the exosomes were isolated promptly and effectively using a novel nanomaterial called ExoQuick. Coomassie brilliant blue staining was used for protein quantification, and the morphology of the exosomes extracted by both methods was visualized by transmission electron microscopy. Exosome marker proteins were detected by Western blot analysis. Two potential hepatoma-associated proteins, tissue transglutaminase 2 (TGM2) and annexin A2, were analyzed.
RESULTS: The exosomes separated by the new extraction assay based on the nanomaterial were disc-shaped, intact vesicles with lipid bilayer membranes. They were approximately 30-100 nm in diameter, which is similar to the diameter of exosomes isolated by the traditional method. The protein concentration of exosomes extracted by the new method was approximately 780 μg/108 cells, and therefore, it was 19 times higher than that of exosomes extracted in the traditional manner. There were differences between the total proteins of Huh-7 cells and the exosomal proteins. Typical exosome proteins, such as the transmembrane protein CD63 and heat shock protein 70, were confirmed. Two potential hepatoma-associated proteins were also identified. TGM2 was first found to exist in the exosomes of human liver cancer cells, but annexin A2 was not secreted into exosomes.
CONCLUSION: The new extraction method based on the nanomaterial is quick and efficient. The cancer-associated protein TGM2 can be secreted through an exosome-mediated non-classical secretion pathway, and it may be a valuable tumor marker.
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