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1
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Guo H, Cai J, Wang X, Wang B, Wang F, Li X, Qu X, Kong X, Gao Y, Wu H, Sun X, Xia Q, Kong X. Prognostic values of a novel multi-mRNA signature for predicting relapse of cholangiocarcinoma. Int J Biol Sci 2020; 16:869-881. [PMID: 32071556 PMCID: PMC7019144 DOI: 10.7150/ijbs.38846] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2019] [Accepted: 11/05/2019] [Indexed: 12/13/2022] Open
Abstract
Cholangiocarcinoma (CCA) is an epithelial cancer and has high death and recurrence rates, current methods cannot satisfy the need for predicting cancer relapse effectively. So, we aimed at conducting a multi-mRNA signature to improve the relapse prediction of CCA. We analyzed mRNA expression profiling in large CCA cohorts from the Gene Expression Omnibus (GEO) database (GSE76297, GSE32879, GSE26566, GSE31370, and GSE45001) and The Cancer Genome Atlas (TCGA) database. The Least absolute shrinkage and selection operator (LASSO) regression model was used to establish a 7-mRNA-based signature that was significantly related to the recurrence-free survival (RFS) in two test series. Based on the 7-mRNA signature, the cohort TCGA patients could be divided into high-risk or low-risk subgroups with significantly different RFS [p < 0.001, hazard ratio (HR): 48.886, 95% confidence interval (CI): 6.226-3.837E+02]. Simultaneously, the prognostic value of the 7-mRNA signature was confirmed in clinical samples of Ren Ji hospital (p < 0.001, HR: 4.558, 95% CI: 1.829-11.357). Further analysis including multivariable and sub-group analyses revealed that the 7-mRNA signature was an independent prognostic value for recurrence of patients with CCA. In conclusion, our results might provide an efficient tool for relapse prediction and were beneficial to individualized management for CCA patients.
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Affiliation(s)
- Han Guo
- Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Jie Cai
- Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Xuan Wang
- Institute of Clinical Immunology, Department of Liver Diseases, Central Laboratory, ShuGuang Hospital Affiliated to Shanghai University of Chinese Traditional Medicine, Shanghai, China
| | - Bingrui Wang
- Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Fang Wang
- Institute of Clinical Immunology, Department of Liver Diseases, Central Laboratory, ShuGuang Hospital Affiliated to Shanghai University of Chinese Traditional Medicine, Shanghai, China
| | - Xiang Li
- Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Xiaoye Qu
- Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Xianming Kong
- Central Laboratory, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Yueqiu Gao
- Institute of Clinical Immunology, Department of Liver Diseases, Central Laboratory, ShuGuang Hospital Affiliated to Shanghai University of Chinese Traditional Medicine, Shanghai, China
| | - Hailong Wu
- Shanghai Key Laboratory for Molecular Imaging, Collaborative Research Center, Shanghai University of Medicine and Health Sciences, Shanghai, China
| | - Xuehua Sun
- Institute of Clinical Immunology, Department of Liver Diseases, Central Laboratory, ShuGuang Hospital Affiliated to Shanghai University of Chinese Traditional Medicine, Shanghai, China
| | - Qiang Xia
- Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Xiaoni Kong
- Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.,Institute of Clinical Immunology, Department of Liver Diseases, Central Laboratory, ShuGuang Hospital Affiliated to Shanghai University of Chinese Traditional Medicine, Shanghai, China
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Rishavy MA, Hallgren KW, Zhang H, Runge KW, Berkner KL. Exon 2 skipping eliminates γ-glutamyl carboxylase activity, indicating a partial splicing defect in a patient with vitamin K clotting factor deficiency. J Thromb Haemost 2019; 17:1053-1063. [PMID: 31009158 PMCID: PMC7181818 DOI: 10.1111/jth.14456] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2018] [Accepted: 03/12/2019] [Indexed: 12/01/2022]
Abstract
Essentials A carboxylase mutation that impairs splicing to delete exon 2 sequences was previously reported. We found that the mutant was inactive for vitamin K-dependent (VKD) protein carboxylation. An incomplete splicing defect likely accounts for VKD clotting activity observed in the patient. The results indicate the importance of proper carboxylase embedment in the membrane for function. BACKGROUND Mutations in the γ-glutamyl carboxylase (GGCX), which is required for vitamin K-dependent (VKD) protein activation, can result in vitamin K clotting factor deficiency (VKCFD1). A recent report described a VKCFD1 patient with a homozygous carboxylase mutation that altered splicing and deleted exon 2 (Δ2GGCX). Only Δ2GGCX RNA was observed in the patient. OBJECTIVES Loss of exon 2 results in the deletion of carboxylase sequences thought to be important for membrane topology and consequent function. Carboxylase activity is required for life, and we therefore tested whether the Δ2GGCX mutant is active. METHODS HEK 293 cells were edited by the use of CRISPR-Cas9 to eliminate endogenous carboxylase. Recombinant wild-type GGCX and recombinant Δ2GGCX were then expressed and tested for carboxylation of the VKD protein factor IX. A second approach was used to monitor carboxylation biochemically, using recombinant carboxylases expressed in insect cells that lack endogenous carboxylase. RESULTS AND CONCLUSIONS Δ2GGCX activity was undetectable in both assays, which is strikingly different from the low levels of carboxylase activity observed with other VKCFD1 mutants. The similarity in clotting function between patients with Δ2GGCX and these mutations must therefore arise from a novel mechanism. Low levels of properly spliced carboxylase RNA that produce full-length protein would not have been observed in the previous study. The results suggest that the splicing defect is incomplete. Δ2GGCX RNA has been detected in normal human liver, and has been designated carboxylase isoform 2; however, Δ2GGCX protein was not observed in normal human liver. The lack of activity and protein expression suggest that isoform 2 is not physiologically relevant to normal VKD protein carboxylation.
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Affiliation(s)
- Mark A Rishavy
- Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine at CWRU, Cleveland Clinic, Cleveland, Ohio
| | - Kevin W Hallgren
- Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine at CWRU, Cleveland Clinic, Cleveland, Ohio
| | - Haitao Zhang
- Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine at CWRU, Cleveland Clinic, Cleveland, Ohio
| | - Kurt W Runge
- Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine at CWRU, Cleveland Clinic, Cleveland, Ohio
| | - Kathleen L Berkner
- Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine at CWRU, Cleveland Clinic, Cleveland, Ohio
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mTOR and ERK regulate VKORC1 expression in both hepatoma cells and hepatocytes which influence blood coagulation. Clin Exp Med 2018; 19:121-132. [PMID: 30306378 DOI: 10.1007/s10238-018-0528-z] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2018] [Accepted: 09/22/2018] [Indexed: 02/08/2023]
Abstract
Deficiency of γ-glutamyl carboxylation of coagulation factors, as evidenced by the elevated level of Des-γ-carboxyl prothrombin (DCP), is a common feature in hepatocellular carcinoma patients. Additionally, treatment of cancer patients with mTOR inhibitors significantly increases hemorrhagic events. However, the underlying mechanisms remain unknown. In the present study, Vitamin K epoxide reductase complex subunit 1 (VKORC1) was found to be significantly down-regulated in clinical hepatoma tissues and most tested hepatoma cell lines. In vitro investigations showed that VKORC1 expression was promoted by p-mTOR at the translational level and repressed by p-ERK at the transcriptional level. By exploring Hras12V transgenic mice, a hepatic tumor model, VKROC1 was significantly down-regulated in hepatic tumors and showed prolonged activated partial prothrombin time (APTT). In vivo investigations further showed that VKORC1 expression was promoted by p-mTOR and repressed by p-ERK in both hepatoma and hepatocytes. Consistently, APTT and prothrombin time were significantly prolonged under the mTOR inhibitor treatment and significantly shortened under the ERK inhibitor treatment. Conclusively, these findings indicate that mTOR and ERK play crucial roles in controlling VKORC1 expression in both hepatoma and hepatocytes, which provides a valuable molecular basis for preventing hemorrhage in clinical therapies.
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Cui SX, Shi WN, Song ZY, Wang SQ, Yu XF, Gao ZH, Qu XJ. Des-gamma-carboxy prothrombin antagonizes the effects of Sorafenib on human hepatocellular carcinoma through activation of the Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways. Oncotarget 2017; 7:36767-36782. [PMID: 27167344 PMCID: PMC5095038 DOI: 10.18632/oncotarget.9168] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2015] [Accepted: 04/23/2016] [Indexed: 12/13/2022] Open
Abstract
Despite significant progress, advanced hepatocellular carcinoma (HCC) remains an incurable disease, and the overall efficacy of targeted therapy by Sorafenib remains moderate. We hypothesized that DCP (des-gamma-carboxy prothrombin), a prothrombin precursor produced in HCC, might be one of the reasons linked to the low efficacy of Sorafenib. We evaluated the efficacy of Sorafenib in HLE and SK-Hep cells, both of which are known DCP-negative HCC cell lines. In the absence of DCP, Sorafenib effectively inhibited the growth of HCC and induced cancer cell apoptosis. In the presence of DCP, HCC was resistant to Sorafenib-induced inhibition and apoptosis, as determined by in vitro assays and in mice xenografted with HLE cells. Molecular analysis of HLE xenografted-nude mice showed that DCP activates the transduction of the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR cascades. DCP might stimulate the formation of compensatory feedback loops in the intricately connected signaling pathways when kinases are targeted by Sorafenib. Our results indicate that DCP antagonizes the inhibitory effects of Sorafenib on HCC through activation of the Ras/Raf/MEK/ERK and Ras/PI3K/Akt/mTOR signaling pathways. Taken together, our findings define a DCP-mediated mechanism of inhibition of Sorafenib in HCC, which is critical for targeting therapy in advanced HCC.
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Affiliation(s)
- Shu-Xiang Cui
- Beijing Key Laboratory of Environmental Toxicology, Department of Toxicology and Sanitary Chemistry, School of Public Health, Capital Medical University, Beijing, China
| | - Wen-Na Shi
- Department of Pharmacology, Capital Medical University School of Basic Medical Sciences, Beijing, China
| | - Zhi-Yu Song
- Department of Pharmacology, Capital Medical University School of Basic Medical Sciences, Beijing, China
| | - Shu-Qing Wang
- Department of Pharmacology, Capital Medical University School of Basic Medical Sciences, Beijing, China
| | - Xin-Feng Yu
- Department of Pharmacology, Capital Medical University School of Basic Medical Sciences, Beijing, China
| | - Zu-Hua Gao
- Department of Pathology, McGill University, Montreal, Quebec, Canada
| | - Xian-Jun Qu
- Department of Pharmacology, Capital Medical University School of Basic Medical Sciences, Beijing, China
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5
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Taniguchi T, Kishi K, Nakagawa T, Tanaka H, Tanaka T, Tomonari T, Okamoto K, Sogabe M, Miyamoto H, Okahisa T, Muguruma N, Kajimoto M, Sagawa I, Takayama T. Poly-(ADP-Ribose) Polymerase-1 Promotes Prothrombin Gene Transcription and Produces Des-Gamma-Carboxy Prothrombin in Hepatocellular Carcinoma. Digestion 2017; 95:242-251. [PMID: 28384634 DOI: 10.1159/000470837] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/06/2017] [Accepted: 03/07/2017] [Indexed: 02/04/2023]
Abstract
BACKGROUND AND AIM Although des-gamma-carboxy prothrombin (DCP) is a well-known tumor marker for hepatocellular carcinoma (HCC), the mechanism of DCP production is unclear. This study aimed to investigate the mechanism how DCP is produced in HCC cells. METHODS Levels of mRNA and DCP were analyzed by real-time polymerase chain reaction and electro-chemiluminescence immunoassay respectively. Secreted alkaline phosphatase (SEAP) expression vectors including deletion mutants of the prothrombin gene promoter were constructed for reporter gene assay. The transcription factors bound to DNA fragments were analyzed by mass spectrometry. An electrophoretic mobility shift assay (EMSA) was performed using a biotin end-labeled DNA. RESULTS The prothrombin mRNA levels in all 5 DCP producing cell lines were appreciably high. However, those in 2 DCP non-producing cell lines were below detectable levels. A SEAP vector with -2985 to +27 showed a very high transcription activity in DCP-producing Huh-1 cells. However, transcription abruptly decreased when the vector with -2955 to +27 was transfected, and then remained at the similar levels with larger deletion mutants, indicating the existence of a cis-element at -2985 to -2955 (31-bp). Mass spectrometry analysis identified the protein that bound to the 31-bp DNA as poly-(ADP-ribose) polymerase-1 (PARP-1). Knockdown of the PARP-1 gene by small interfering RNA in Huh-1 cells induced marked inhibition of prothrombin gene transcription. The EMSA clearly showed that PARP-1 specifically binds to the 31-bp DNA fragment in the prothrombin gene promoter. CONCLUSIONS Our data suggest that PARP-1 activates prothrombin gene transcription and that the excessive prothrombin gene transcription induces DCP production in DCP-producing HCC cells.
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MESH Headings
- Alkaline Phosphatase/metabolism
- Biomarkers/metabolism
- Biomarkers, Tumor/genetics
- Biomarkers, Tumor/metabolism
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/pathology
- Cell Line, Tumor
- Cell Survival
- Electrophoretic Mobility Shift Assay
- Gene Expression Regulation, Neoplastic
- Gene Knockdown Techniques
- Genes, Reporter
- Humans
- Immunoenzyme Techniques
- Liver Neoplasms/genetics
- Liver Neoplasms/pathology
- Mass Spectrometry
- Poly (ADP-Ribose) Polymerase-1/genetics
- Poly (ADP-Ribose) Polymerase-1/metabolism
- Protein Precursors/genetics
- Protein Precursors/metabolism
- Prothrombin/genetics
- Prothrombin/metabolism
- RNA Interference
- RNA, Messenger/metabolism
- RNA, Small Interfering/metabolism
- Real-Time Polymerase Chain Reaction
- Transcription, Genetic
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Affiliation(s)
- Tatsuya Taniguchi
- Department of Gastroenterology and Oncology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan
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Dasi MA, Gonzalez-Conejero R, Izquierdo S, Padilla J, Garcia JL, Garcia-Barberá N, Argilés B, de la Morena-Barrio ME, Hernández-Sánchez JM, Hernández-Rivas JM, Vicente V, Corral J. Uniparental disomy causes deficiencies of vitamin K-dependent proteins. J Thromb Haemost 2016; 14:2410-2418. [PMID: 27681307 DOI: 10.1111/jth.13517] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2016] [Accepted: 08/08/2016] [Indexed: 08/31/2023]
Abstract
Essentials Vitamin K-dependent coagulant factor deficiency (VKCFD) is a rare autosomal recessive disorder. We describe a case of inherited VKCFD due to uniparental disomy. The homozygous mutation caused the absence of GGCX isoform 1 and overexpression of Δ2GGCX. Hepatic and non-hepatic vitamin K-dependent proteins must be assayed to monitor VKCFD treatment. SUMMARY Background Inherited deficiency of all vitamin K-dependent coagulant factors (VKCFD) is a rare autosomal recessive disorder caused by mutations in the γ-glutamyl carboxylase gene (GGCX) or the vitamin K epoxide reductase gene (VKORC1), with great heterogeneity in terms of both clinical presentation and response to treatment. Objective To characterize the molecular basis of VKCFD in a Spanish family. Methods and Results Sequencing of candidate genes, comparative genomic hybridization and massive sequencing identified a new mechanism causing VKCFD in the proband. Uniparental disomy (UPD) of chromosome 2 caused homozygosity of a mutation (c.44-1G>A) resulting in aberrant GGCX splicing. This change contributed to absent expression of the mRNA coding for the full-length protein, and to four-fold overexpression of the smaller mRNA isoform lacking exon 2 (Δ2GGCX). Δ2GGCX might be responsible for two unexpected clinical observations in the patient: (i) increased plasma osteocalcin levels following vitamin K1 supplementation; and (ii) a mild non-bleeding phenotype. Conclusions Our study identifies a new autosomal disease, VKCFD1, caused by UPD. These data suggest that the Δ2GGCX isoform may retain enzymatic activity, and strongly encourage the evaluation of both hepatic and non-hepatic vitamin K-dependent proteins to assess differing responses to vitamin K supplementation in VKCFD patients.
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Affiliation(s)
- M A Dasi
- Unidad de Hematología Pediátrica, Hospital Universitario y Politécnico La Fe, Valencia, Spain
| | | | - S Izquierdo
- Unidad de Hematología Pediátrica, Hospital Universitario y Politécnico La Fe, Valencia, Spain
| | - J Padilla
- Centro Regional de Hemodonación, Universidad de Murcia-IMIB, Murcia, Spain
| | - J L Garcia
- Centro de Investigación del Cáncer-Universidad de Salamanca-CSIC, Salamanca, Spain
| | - N Garcia-Barberá
- Centro Regional de Hemodonación, Universidad de Murcia-IMIB, Murcia, Spain
| | - B Argilés
- Unidad de Hematología Pediátrica, Hospital Universitario y Politécnico La Fe, Valencia, Spain
| | - M E de la Morena-Barrio
- Centro Regional de Hemodonación, Universidad de Murcia-IMIB, Murcia, Spain
- Grupo CB15/00055 del Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain
| | | | - J M Hernández-Rivas
- Centro de Investigación del Cáncer-Universidad de Salamanca-CSIC, Salamanca, Spain
| | - V Vicente
- Centro Regional de Hemodonación, Universidad de Murcia-IMIB, Murcia, Spain
- Grupo CB15/00055 del Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain
| | - J Corral
- Centro Regional de Hemodonación, Universidad de Murcia-IMIB, Murcia, Spain
- Grupo CB15/00055 del Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain
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Jinghe X, Mizuta T, Ozaki I. Vitamin K and hepatocellular carcinoma: The basic and clinic. World J Clin Cases 2015; 3:757-64. [PMID: 26380822 PMCID: PMC4568524 DOI: 10.12998/wjcc.v3.i9.757] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/25/2015] [Revised: 06/20/2015] [Accepted: 07/24/2015] [Indexed: 02/05/2023] Open
Abstract
Vitamin K (VK), which was originally identified as a cofactor involved in the production of functional coagulation factors in the liver, has been shown to be involved in various aspects of physiological and pathological events, including bone metabolism, cardiovascular diseases and tumor biology. The mechanisms and roles of VK are gradually becoming clear. Several novel enzymes involved in the VK cycle were identified and have been shown to be linked to tumorigenesis. The VKs have been shown to suppress liver cancer cell growth through multiple signaling pathways via the transcription factors and protein kinases. A VK2 analog was applied to the chemoprevention of hepatocellular carcinoma (HCC) recurrence after curative therapy and was shown to have beneficial effects, both in the suppression of HCC recurrence and in patient survival. Although a large scale randomized control study failed to demonstrate the suppression of HCC recurrence, a meta-analysis suggested a beneficial effect on the long-term survival of HCC patients. However, the beneficial effects of VK administration alone were not sufficient to prevent or treat HCC in clinical settings. Thus its combination with other anti-cancer reagents and the development of more potent novel VK derivatives are the focus of ongoing research which seeks to achieve satisfactory therapeutic effects against HCC.
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8
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Yamashita YI, Shirabe K, Aishima S, Maehara Y. Predictors of Microvascular Invasion in Hepatocellular Carcinoma. Dig Dis 2015; 33:655-60. [PMID: 26398341 DOI: 10.1159/000438475] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
This chapter covers a range of important topics in the evaluation of the microvascular invasion (MVI) in hepatocellular carcinoma (HCC) before treatment. The malignant potential of HCC is reflected by the types of MVI such as portal venous (vp), hepatic vein (vv) or bile duct (b) infiltration. The identification of the type of MVI in HCC has a key role in decisions regarding the effective treatment of HCC. Here, we describe the possible and important predictors of MVI in HCC.
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Affiliation(s)
- Yo-Ichi Yamashita
- Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
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Gla-rich protein is a potential new vitamin K target in cancer: evidences for a direct GRP-mineral interaction. BIOMED RESEARCH INTERNATIONAL 2014; 2014:340216. [PMID: 24949434 PMCID: PMC4052551 DOI: 10.1155/2014/340216] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/24/2014] [Accepted: 04/08/2014] [Indexed: 12/16/2022]
Abstract
Gla-rich protein (GRP) was described in sturgeon as a new vitamin-K-dependent protein (VKDP) with a high density of Gla residues and associated with ectopic calcifications in humans. Although VKDPs function has been related with γ-carboxylation, the Gla status of GRP in humans is still unknown. Here, we investigated the expression of recently identified GRP spliced transcripts, the γ-carboxylation status, and its association with ectopic calcifications, in skin basal cell and breast carcinomas. GRP-F1 was identified as the predominant splice variant expressed in healthy and cancer tissues. Patterns of γ-carboxylated GRP (cGRP)/undercarboxylated GRP (ucGRP) accumulation in healthy and cancer tissues were determined by immunohistochemistry, using newly developed conformation-specific antibodies. Both GRP protein forms were found colocalized in healthy tissues, while ucGRP was the predominant form associated with tumor cells. Both cGRP and ucGRP found at sites of microcalcifications were shown to have in vitro calcium mineral-binding capacity. The decreased levels of cGRP and predominance of ucGRP in tumor cells suggest that GRP may represent a new target for the anticancer potential of vitamin K. Also, the direct interaction of cGRP and ucGRP with BCP crystals provides a possible mechanism explaining GRP association with pathological mineralization.
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10
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Matsubara M, Shiraha H, Kataoka J, Iwamuro M, Horiguchi S, Nishina SI, Takaoka N, Uemura M, Takaki A, Nakamura S, Kobayashi Y, Nouso K, Yamamoto K. Des-γ-carboxyl prothrombin is associated with tumor angiogenesis in hepatocellular carcinoma. J Gastroenterol Hepatol 2012; 27:1602-1608. [PMID: 22554292 DOI: 10.1111/j.1440-1746.2012.07173.x] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
BACKGROUND AND AIM Hepatocellular carcinoma (HCC) is a hypervascular tumor, and angiogenesis plays an important role in its development. Previously, we demonstrated that des-γ-carboxyl prothrombin (DCP) promotes both cell proliferation and migration of human umbilical vein endothelial cells (HUVECs) by inducing the autophosphorylation of kinase insert domain receptor (KDR). In the present study, DCP-associated tumor angiogenesis was assessed by comparing hypovascular and common hypervascular HCC. METHODS The solitary HCCs of 827 patients were classified into two groups according to the tumor density at the arterial phase of a dynamic computed tomography scan; the initial clinical data of patients with the hyper- and hypovascular types were compared. The HCC tissues from 95 tumors were analyzed by immunohistochemical staining for DCP and phosphorylated KDR, and intratumoral microvessel density (MVD) was analyzed to evaluate microvessel angiogenesis. RESULTS The serum DCP levels (320 ± 3532 mAU/mL) and tumor size (18.4 ± 9.0 mm) of patients with hypervascular HCC were significantly greater than those with hypovascular HCC (38.7 ± 80 mAU/mL and 14.6 ± 5.2 mm, P < 0.001). Immunohistochemical analysis revealed that the expressions of DCP and phospho-KDR were significantly greater in hypervascular HCC (71.4% and 31.0%, respectively) than in hypovascular HCC (7.6% and 5.7%, respectively). Intratumoral MVD was significantly correlated with DCP (r = 0.48, P < 0.0001). CONCLUSIONS des-γ-carboxyl prothrombin production is associated with tumor angiogenesis in HCC.
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MESH Headings
- Adult
- Aged
- Aged, 80 and over
- Biomarkers/blood
- Biomarkers/metabolism
- Biomarkers, Tumor/blood
- Biomarkers, Tumor/metabolism
- Carbon-Carbon Ligases/genetics
- Carbon-Carbon Ligases/metabolism
- Carcinoma, Hepatocellular/blood
- Carcinoma, Hepatocellular/blood supply
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/metabolism
- Carcinoma, Hepatocellular/pathology
- Cell Line, Tumor
- Culture Media, Conditioned/metabolism
- Female
- Human Umbilical Vein Endothelial Cells/metabolism
- Humans
- Immunohistochemistry
- Liver Neoplasms/blood
- Liver Neoplasms/blood supply
- Liver Neoplasms/genetics
- Liver Neoplasms/metabolism
- Liver Neoplasms/pathology
- Male
- Middle Aged
- Neovascularization, Pathologic
- Phosphorylation
- Protein Precursors/blood
- Protein Precursors/metabolism
- Prothrombin/metabolism
- Tomography, X-Ray Computed
- Transfection
- Tumor Burden
- Up-Regulation
- Vascular Endothelial Growth Factor Receptor-2/metabolism
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Affiliation(s)
- Minoru Matsubara
- Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
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11
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Yue P, Gao ZH, Xue X, Cui SX, Zhao CR, Yuan Y, Yin Z, Inagaki Y, Kokudo N, Tang W, Qu XJ. Des-γ-carboxyl prothrombin induces matrix metalloproteinase activity in hepatocellular carcinoma cells by involving the ERK1/2 MAPK signalling pathway. Eur J Cancer 2011; 47:1115-24. [PMID: 21349701 DOI: 10.1016/j.ejca.2011.01.017] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2010] [Revised: 01/18/2011] [Accepted: 01/26/2011] [Indexed: 12/22/2022]
Abstract
Des-γ-carboxy prothrombin (DCP), an aberrant prothrombin produced by hepatocellular carcinoma (HCC) cells, has been shown to be associated with the biological malignant potential of HCC. The aim of this study was to evaluate the effect of DCP on HCC cell growth and metastasis, and to explore the underlying molecular mechanisms. DCP significantly stimulated HCC cell growth, as measured by cell counting kit-8 assay. Transwell chamber assay showed that DCP increased HCC cell migration through reconstituted extracellular matrix (Matrigel). Gelatin zymography assay and Western blot analysis demonstrated that DCP increased the secretion and expression of matrix metalloproteinase (MMP)-2 and MMP-9 in the supernatant of cultured HCC cells and on tumour cell membranes. DCP was found to bind to the cell surface receptor Met, resulting in Met phosphorylation and subsequent activation of the epidermal growth factor receptor (EGFR). Western blot analysis demonstrated that DCP stimulated a sequential kinase phosphorylation cascade including ERK1/2, MEK1/2 and c-Raf, indicating activation of the extracellular signal-regulated kinase/mitogen activated protein kinase (ERK1/2 MAPK) signalling pathway. Furthermore, blocking ERK1/2 MAPK activation with ERK1/2 inhibitor PD98059 essentially abolished the DCP-induced MMP-2 and MMP-9 activity, confirming the signalling pathway of DCP stimulation. Taken together, these results suggested that DCP stimulates HCC growth and promotes HCC metastasis by increasing the activity of MMP-2 and MMP-9 through activation of the ERK1/2 MAPK signalling pathway.
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Affiliation(s)
- Pan Yue
- Department of Pharmacology, School of Pharmaceutical Sciences, Shandong University, Jinan, China
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Inagaki Y, Tang W, Makuuchi M, Hasegawa K, Sugawara Y, Kokudo N. Clinical and molecular insights into the hepatocellular carcinoma tumour marker des-γ-carboxyprothrombin. Liver Int 2011; 31:22-35. [PMID: 20874725 DOI: 10.1111/j.1478-3231.2010.02348.x] [Citation(s) in RCA: 98] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Des-γ-carboxyprothrombin (DCP) is known as a tumour marker for hepatocellular carcinoma (HCC). Various tumour markers have been developed for serological diagnosis of cancers, including HCC, in order to increase the survival rate of cancer patients. The currently recommended combined testing of DCP and α-fetoprotein (AFP) or Lens culinaris agglutinin-reactive fraction of α-fetoprotein has been established to diagnose HCC. This combined testing using several tumour markers helps to increase the sensitivity of diagnosis of HCC, thus significantly increasing the clinical usefulness of DCP. The excessive production of DCP may be related to worse tumour behaviour, such as the presence of vascular invasion and intrahepatic metastasis of HCC cells. A high level of DCP was suggested to be useful as one of the factors in new recipient selection criteria of liver transplantation. The clinical use of DCP, therefore, might play a vital role in predicting tumour behaviour in patients with HCC. That said, the basic mechanism of DCP production has not been fully clarified. Various factors such as vitamin K(2) and γ-glutamyl carboxylase may contribute to the production of DCP and have a complex relationship. Moreover, recent studies have revealed that DCP functions as a growth factor and might play significant roles in cancer progression. Thus, DCP represents a potential target of drug discovery to establish new chemotherapeutic strategy for HCC. However, various issues have to be resolved to construct a novel therapy for HCC-targeting DCP. Innovation is required to make further progress in examining DCP.
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Affiliation(s)
- Yoshinori Inagaki
- Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan
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Ueda N, Shiraha H, Fujikawa T, Takaoka N, Nakanishi Y, Suzuki M, Matsuo N, Tanaka S, Nishina SI, Uemura M, Takaki A, Shiratori Y, Yamamoto K. Exon 2 deletion splice variant of gamma-glutamyl carboxylase causes des-gamma-carboxy prothrombin production in hepatocellular carcinoma cell lines. Mol Oncol 2008; 2:241-9. [PMID: 19383345 DOI: 10.1016/j.molonc.2008.06.004] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2008] [Revised: 06/11/2008] [Accepted: 06/15/2008] [Indexed: 01/02/2023] Open
Abstract
Using GGCX gene-specific real-time PCR, exon 2 deletion splice variant of vitamin K-dependent gamma-glutamyl carboxylase (GGCX) mRNA was identified in HCC cell lines. Expressions of wild type and exon 2 deletion variant of GGCX were analyzed with relevance to DCP production in HCC cell lines. Hep3B, HepG2, HuH1, HuH7, and PLC/PRF/5 produced DCP, while SK-Hep-1, HLE, HLF, and JHH1 produced no detectable level of DCP. DCP-producing cells expressed exon 2 deletion variant of GGCX mRNA and protein, while DCP-negative cells expressed no detectable level of exon 2 deletion variant of GGCX. These results suggest that exon 2 deletion splice variant of GGCX causes dysfunction of GGCX enzyme activity resulting in DCP production in HCC cell lines.
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Affiliation(s)
- Naoki Ueda
- Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
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