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Wyżewski Z, Gregorczyk-Zboroch KP, Mielcarska MB, Świtlik W, Niedzielska A. Bid Protein: A Participant in the Apoptotic Network with Roles in Viral Infections. Int J Mol Sci 2025; 26:2385. [PMID: 40141030 PMCID: PMC11942203 DOI: 10.3390/ijms26062385] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2025] [Revised: 03/01/2025] [Accepted: 03/04/2025] [Indexed: 03/28/2025] Open
Abstract
The BH3-interacting domain death agonist (Bid), a proapoptotic signaling molecule of the B-cell lymphoma 2 (Bcl-2) family, is a key regulator of mitochondrial outer membrane (MOM) permeability. Uniquely positioned at the intersection of extrinsic and intrinsic apoptosis pathways, Bid links death receptor signaling to the mitochondria-dependent cascade and can also be activated by endoplasmic reticulum (ER) stress. In its active forms, cleaved Bid (cBid) and truncated Bid (tBid), it disrupts MOM integrity via Bax/Bak-dependent and independent mechanisms. Apoptosis plays a dual role in viral infections, either promoting or counteracting viral propagation. Consequently, viruses modulate Bid signaling to favor their replication. The deregulation of Bid activity contributes to oncogenic transformation, inflammation, immunosuppression, neurotoxicity, and pathogen propagation during various viral infections. In this work, we explore Bid's structure, function, activation processes, and mitochondrial targeting. We describe its role in apoptosis induction and its involvement in infections with multiple viruses. Additionally, we discuss the therapeutic potential of Bid in antiviral strategies. Understanding Bid's signaling pathways offers valuable insights into host-virus interactions and the pathogenesis of infections. This knowledge may facilitate the development of novel therapeutic approaches to combat virus-associated diseases effectively.
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Affiliation(s)
- Zbigniew Wyżewski
- Institute of Biological Sciences, Cardinal Stefan Wyszynski University in Warsaw, Dewajtis 5, 01-815 Warsaw, Poland
| | - Karolina Paulina Gregorczyk-Zboroch
- Division of Immunology, Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences—SGGW, Ciszewskiego 8, 02-786 Warsaw, Poland; (K.P.G.-Z.); (M.B.M.); (A.N.)
| | - Matylda Barbara Mielcarska
- Division of Immunology, Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences—SGGW, Ciszewskiego 8, 02-786 Warsaw, Poland; (K.P.G.-Z.); (M.B.M.); (A.N.)
| | - Weronika Świtlik
- Centre for Advanced Materials and Technologies, Warsaw University of Technology, Poleczki 19, 02-822 Warsaw, Poland;
| | - Adrianna Niedzielska
- Division of Immunology, Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences—SGGW, Ciszewskiego 8, 02-786 Warsaw, Poland; (K.P.G.-Z.); (M.B.M.); (A.N.)
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Mohamed HR, Hamed MM, El-Wakil EA, Okasha H. GC-MS analysis, anti-inflammatory and anti-proliferative properties of the aerial parts of three Mesembryanthemum spp.. Toxicol Rep 2024; 13:101829. [PMID: 39735355 PMCID: PMC11681886 DOI: 10.1016/j.toxrep.2024.101829] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2024] [Revised: 11/13/2024] [Accepted: 11/22/2024] [Indexed: 12/31/2024] Open
Abstract
Background Due to their variability and safety, widespread research on phytochemicals continually encourages researchers to study various plants for their potential health benefits. Objectives This study aims to explore the phytochemical constituents of the aerial parts of three Mesembryanthemum spp.; M. nodiflorum, M. forsskaolii, and M. cordifolium existed in Egyptian nature using GC-MS analysis and studying their different biological activities in correlation to computational analysis. Methods Investigation of in vitro anti-inflammatory and anticancer activities and in silico studies of identified major compounds on VEGFR. Results: Thirty-three compounds were identified, octadecanoic acid, 2, 3-dihydroxypropyl ester, and 1H-Indene, 1-hexadecyl-2, 3-dihydro are the common compounds in the three extracts with different percentages. M. forsskaolii is the most extract with diverse phytoconstituents showing significant anticancer properties against the CACO2 cells with IC50 value equal to 31.78 µg/mL. Nevertheless, all extracts showed potent anti-inflammatory activity at high concentrations (500 µg/mL). M. nodiflorum, M. forsskaolii, and M. cordifolium had IC50 on HepG2 cells equal to 73.64, 88.18, and 87.82 µg/mL. Molecular findings showed the three extracts had distinct effects on apoptosis modulation in HepG2 cells. Conclusion The findings suggest that the studied extracts had potential therapeutic properties as anti-inflammatory and anticancer agents, supported by an in-silico interaction study.
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Affiliation(s)
- Heba R. Mohamed
- Department of Medicinal Chemistry, Theodor Bilharz Research Institute, Kornish El-Nile, Warrak El-Hadar, Giza 12411, Egypt
| | - Manal M. Hamed
- Department of Medicinal Chemistry, Theodor Bilharz Research Institute, Kornish El-Nile, Warrak El-Hadar, Giza 12411, Egypt
| | - Eman A. El-Wakil
- Department of Medicinal Chemistry, Theodor Bilharz Research Institute, Kornish El-Nile, Warrak El-Hadar, Giza 12411, Egypt
| | - Hend Okasha
- Department of Biochemistry and Molecular Biology, Theodor Bilharz Research Institute, Kornish El-Nile, Warrak El-Hadar, Giza 12411, Egypt
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Shteinfer-Kuzmine A, Verma A, Bornshten R, Ben Chetrit E, Ben-Ya'acov A, Pahima H, Rubin E, Mograbi Y, Shteyer E, Shoshan-Barmatz V. Elevated serum mtDNA in COVID-19 patients is linked to SARS-CoV-2 envelope protein targeting mitochondrial VDAC1, inducing apoptosis and mtDNA release. Apoptosis 2024; 29:2025-2046. [PMID: 39375263 PMCID: PMC11550248 DOI: 10.1007/s10495-024-02025-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/23/2024] [Indexed: 10/09/2024]
Abstract
Mitochondria dysfunction is implicated in cell death, inflammation, and autoimmunity. During viral infections, some viruses employ different strategies to disrupt mitochondria-dependent apoptosis, while others, including SARS-CoV-2, induce host cell apoptosis to facilitate replication and immune system modulation. Given mitochondrial DNAs (mtDNA) role as a pro-inflammatory damage-associated molecular pattern in inflammatory diseases, we examined its levels in the serum of COVID-19 patients and found it to be high relative to levels in healthy donors. Furthermore, comparison of serum protein profiles between healthy individuals and SARS-CoV-2-infected patients revealed unique bands in the COVID-19 patients. Using mass spectroscopy, we identified over 15 proteins, whose levels in the serum of COVID-19 patients were 4- to 780-fold higher. As mtDNA release from the mitochondria is mediated by the oligomeric form of the mitochondrial-gatekeeper-the voltage-dependent anion-selective channel 1 (VDAC1)-we investigated whether SARS-CoV-2 protein alters VDAC1 expression. Among the three selected SARS-CoV-2 proteins, small envelope (E), nucleocapsid (N), and accessory 3b proteins, the E-protein induced VDAC1 overexpression, VDAC1 oligomerization, cell death, and mtDNA release. Additionally, this protein led to mitochondrial dysfunction, as evidenced by increased mitochondrial ROS production and cytosolic Ca2+ levels. These findings suggest that SARS-CoV-2 E-protein induces mitochondrial dysfunction, apoptosis, and mtDNA release via VDAC1 modulation. mtDNA that accumulates in the blood activates the cGAS-STING pathway, triggering inflammatory cytokine and chemokine expression that contribute to the cytokine storm and tissue damage seen in cases of severe COVID-19.
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Affiliation(s)
| | - Ankit Verma
- National Institute for Biotechnology in the Negev, Beer-Sheva, Israel
- Department of Life Sciences, Ben-Gurion University of the Negev, 84105, Beer-Sheva, Israel
| | - Rut Bornshten
- The Shraga Segal Dept. of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, 84105, Beer-Sheva, Israel
| | - Eli Ben Chetrit
- Infectious Diseases Unit, Shaare Zedek Medical Center, Hebrew University School of Medicine, Jerusalem, Israel
| | - Ami Ben-Ya'acov
- Shaare Zedek Medical Center, The Juliet Keidan Institute of Paediatric Gastroenterology, Jerusalem, Israel
| | - Hadas Pahima
- Department of Life Sciences, Ben-Gurion University of the Negev, 84105, Beer-Sheva, Israel
| | - Ethan Rubin
- The Shraga Segal Dept. of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, 84105, Beer-Sheva, Israel
- Shaare Zedek Medical Center, The Juliet Keidan Institute of Paediatric Gastroenterology, Jerusalem, Israel
| | | | - Eyal Shteyer
- Shaare Zedek Medical Center, The Juliet Keidan Institute of Paediatric Gastroenterology, Jerusalem, Israel
| | - Varda Shoshan-Barmatz
- National Institute for Biotechnology in the Negev, Beer-Sheva, Israel.
- Department of Life Sciences, Ben-Gurion University of the Negev, 84105, Beer-Sheva, Israel.
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Reffai A, Hori M, Adusumilli R, Bermudez A, Bouzoubaa A, Pitteri S, Bennani Mechita M, Mallick P. A Proteomic Analysis of Nasopharyngeal Carcinoma in a Moroccan Subpopulation. Cancers (Basel) 2024; 16:3282. [PMID: 39409902 PMCID: PMC11476039 DOI: 10.3390/cancers16193282] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2024] [Revised: 09/14/2024] [Accepted: 09/19/2024] [Indexed: 10/20/2024] Open
Abstract
BACKGROUND Nasopharyngeal carcinoma (NPC) is a distinct cancer of the head and neck that is highly prevalent in Southeast Asia and North Africa. Though an extensive analysis of environmental and genetic contributors has been performed, very little is known about the proteome of this disease. A proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissues can provide valuable information on protein expression and molecular patterns for both increasing our understanding of the disease and for biomarker discovery. To date, very few NPC proteomic studies have been performed, and none focused on patients from Morocco and North Africa. METHODS Label-free Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) was used to perform a proteomic analysis of FFPE tissue samples from a cohort of 41 NPC tumor samples of Morocco and North Africa origins. The LC-MS/MS data from this cohort were analyzed alongside 21 healthy controls using MaxQuant 2.4.2.0. A differential expression analysis was performed using the MSstats package in R. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotations were carried out using the DAVID bioinformatic tool. RESULTS 3341 proteins were identified across our NPC cases, revealing three main clusters and five DEPs with prognostic significance. The sex disparity of NPC was investigated from a proteomic perspective in which 59 DEPs were found between males and females, with significantly enriched terms associated with the immune response and gene expression. Furthermore, 26 DEPs were observed between patients with early and advanced stages of NPC with a significant cluster related to the immune response, implicating up-regulated DEPs such as IGHA, IGKC, and VAT1. Across both datasets, 6532 proteins were quantified between NPC patients and healthy controls. Among them, 1507 differentially expressed proteins (DEPs) were observed. GO and KEGG pathway analyses showed enriched terms of DEPs related to increased cellular activity, cell proliferation, and survival. PI3K and MAPK proteins as well as RAC1 BCL2 and PPIA were found to be overexpressed between cancer tissues and healthy controls. EBV infection was also one of the enriched pathways implicating its latent genes like LMP1 and LMP2 that activate several proteins and signaling pathways including NF-Kappa B, MAPK, and JAK-STAT pathways. CONCLUSION Our findings unveil the proteomic landscape of NPC for the first time in the Moroccan population. These studies additionally may provide a foundation for identifying potential biomarkers. Further research is still needed to help develop tools for the early diagnosis and treatment of NPC in Moroccan and North African populations.
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Affiliation(s)
- Ayman Reffai
- Intelligent Automation and BioMed Genomics Laboratory, Biology Department, Faculty of Sciences and Techniques of Tangier, Abdelmalek Essaadi University-Tetouan, Tangier 90000, Morocco
- Canary Center for Cancer Early Detection, School of Medicine, Stanford University, Stanford, CA 94305, USA
| | - Michelle Hori
- Canary Center for Cancer Early Detection, School of Medicine, Stanford University, Stanford, CA 94305, USA
| | - Ravali Adusumilli
- Canary Center for Cancer Early Detection, School of Medicine, Stanford University, Stanford, CA 94305, USA
| | - Abel Bermudez
- Canary Center for Cancer Early Detection, School of Medicine, Stanford University, Stanford, CA 94305, USA
| | | | - Sharon Pitteri
- Canary Center for Cancer Early Detection, School of Medicine, Stanford University, Stanford, CA 94305, USA
| | - Mohcine Bennani Mechita
- Intelligent Automation and BioMed Genomics Laboratory, Biology Department, Faculty of Sciences and Techniques of Tangier, Abdelmalek Essaadi University-Tetouan, Tangier 90000, Morocco
| | - Parag Mallick
- Canary Center for Cancer Early Detection, School of Medicine, Stanford University, Stanford, CA 94305, USA
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Elizalde MM, Sevic I, González López Ledesma MM, Campos RH, Barbini L, Flichman DM. Human hepatocytes apoptosis induced by replication of hepatitis B virus subgenotypes F1b and F4: Role of basal core promoter and preCore mutations. Virology 2018; 513:160-167. [DOI: 10.1016/j.virol.2017.10.016] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2017] [Revised: 10/03/2017] [Accepted: 10/19/2017] [Indexed: 02/06/2023]
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Wang G, Dong F, Xu Z, Sharma S, Hu X, Chen D, Zhang L, Zhang J, Dong Q. MicroRNA profile in HBV-induced infection and hepatocellular carcinoma. BMC Cancer 2017; 17:805. [PMID: 29191172 PMCID: PMC5709924 DOI: 10.1186/s12885-017-3816-1] [Citation(s) in RCA: 60] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2016] [Accepted: 11/22/2017] [Indexed: 02/07/2023] Open
Abstract
Background MicroRNAs (miRNAs) exhibit essential regulatory functions related to cell growth, apoptosis, development and differentiation. Dysregulated expression of miRNAs is associated with a wide variety of human diseases. As such miRNA signatures are valuable as biomarkers for disease and for making treatment decisions. Hepatitis B virus (HBV) is a major risk factor for hepatocellular carcinoma (HCC). Here we screened for miRNAs in chronic HBV associated HCC. Methods To determine the miRNAs in HCC occurrence associated with HBV infection, we analyzed global miRNA expression profiles in 12 pairs of HCC and adjacent matched non-HCC tissues from HBV-positive and HBV-negative patients using microarray analyses. The microarray result was validated by real-time PCR in 32 HBV-positive and 24 HBV-negative patient HCC samples. The potential candidate target genes of the miRNAs were predicted by miRWalk software. Genes simultaneously predicted as targets by two or more miRNAs were subjected to GO and KEGG pathway analysis. The miRNA regulatory network analysis was performed using the Ingenuity Pathway Analysis (IPA) software. Results Eight miRNAs (miR-223, miR-98, miR-15b, miR-199a-5p, miR-19b, miR-22, miR-451, and miR-101) were involved in HBV-unrelated HCC, 5 miRNAs (miR-98, miR-375, miR-335, miR-199a-5p, and miR-22) were involved in HBV infection, and 7 miRNAs (miR-150, miR-342-3p, miR-663, miR-20b, miR-92a-3p, miR-376c-3p and miR-92b) were specifically altered in HBV-related HCC. Gene Ontology and KEGG analyses predict that these HBV-related HCC miRNAs are involved in the regulation of: transcription, RNA polymerase II promoter, phosphorylation of proteins through MAPK signaling pathway, focal adhesion, and actin cytoskeleton. IPA analysis also suggest that these miRNAs act on AGO2, TP53, CCND1, and 11 other genes that significantly influence HCC occurrence and HBV infection. Conclusion Our data indicates that the unique 7 miRNAs expression signature could be involved in the development HBV- related HCC. Electronic supplementary material The online version of this article (10.1186/s12885-017-3816-1) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Guanyu Wang
- Department of General Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
| | - Fulu Dong
- Institutes of Biology and Medical Sciences, Soochow University, Soochow, Jiangsu, China
| | - Zhiyao Xu
- Key Lab of Biomedical Research Center, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
| | - Sherven Sharma
- David Geffen School of Medicine at UCLA, and the Department of Veterans Affairs, Los Angeles, CA, USA
| | - Xiaotong Hu
- Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, Zhejiang, China
| | - Dafang Chen
- Key Lab of Biomedical Research Center, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
| | - Lumin Zhang
- Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, Zhejiang, China
| | - Jinping Zhang
- Institutes of Biology and Medical Sciences, Soochow University, Soochow, Jiangsu, China.
| | - Qinghua Dong
- Key Lab of Biomedical Research Center, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China. .,Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education, Hangzhou, China.
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Abstract
Hepatocellular carcinoma (HCC) is a major health problem. In human hepatocarcinogenesis, the balance between cell death and proliferation is deregulated, tipping the scales for a situation where antiapoptotic signals are overpowering the death-triggering stimuli. HCC cells harbor a wide variety of mutations that alter the regulation of apoptosis and hence the response to chemotherapeutical drugs, making them resistant to the proapoptotic signals. Considering all these modifications found in HCC cells, therapeutic approaches need to be carefully studied in order to specifically target the antiapoptotic signals. This review deals with the recent relevant contributions reporting molecular alterations for HCC that lead to a deregulation of apoptosis, as well as the challenge of death-inducing chemotherapeutics in current HCC treatment.
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Affiliation(s)
- Joaquim Moreno-Càceres
- Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain.,Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain
| | - Isabel Fabregat
- Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain.,Department of Physiological Sciences II, University of Barcelona, Spain.,Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain.,Department of Physiological Sciences II, University of Barcelona, Spain
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Ayub A, Ashfaq UA, Haque A. HBV induced HCC: major risk factors from genetic to molecular level. BIOMED RESEARCH INTERNATIONAL 2013; 2013:810461. [PMID: 23991421 PMCID: PMC3749539 DOI: 10.1155/2013/810461] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/18/2013] [Accepted: 07/09/2013] [Indexed: 12/15/2022]
Abstract
Hepatocellular carcinoma (HCC) is a deadly and emerging disease leading to death in Asian countries. High hepatitis B virus (HBV) load and chronic hepatitis B (CHB) infection increase the risk of developing HCC. HBV is a DNA virus that can integrate DNA into host genome thereby increase the yield of transactivator protein HBxAg that may deregulate many pathways involving in metabolism of cells. Several monogenic and polygenic risk factors are also involved in HCC development. This review summarizes the mechanism involved in HCC development and discusses some promising therapies to make HCC curative.
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Affiliation(s)
- Ambreen Ayub
- Department of Bioinformatics and Biotechnology, Government College University Faisalabad (GCUF), Faisalabad 38000, Pakistan
| | - Usman Ali Ashfaq
- Department of Bioinformatics and Biotechnology, Government College University Faisalabad (GCUF), Faisalabad 38000, Pakistan
| | - Asma Haque
- Department of Bioinformatics and Biotechnology, Government College University Faisalabad (GCUF), Faisalabad 38000, Pakistan
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Potent killing of HBV-related hepatocellular carcinoma by a chimeric protein of anti-HBsAg single-chain antibody and truncated Bid. Biomaterials 2013; 34:4880-9. [PMID: 23562050 DOI: 10.1016/j.biomaterials.2013.03.046] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2013] [Accepted: 03/15/2013] [Indexed: 01/18/2023]
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Zhang CZY, Cao Y, Yun JP, Chen GG, Lai PBS. Increased expression of ZBP-89 and its prognostic significance in hepatocellular carcinoma. Histopathology 2012; 60:1114-24. [PMID: 22372401 DOI: 10.1111/j.1365-2559.2011.04136.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
AIMS ZBP-89 plays a role in cell growth and death. Its expression in hepatocellular carcinoma (HCC) is not well documented. This study aimed to analyse ZBP-89 expression in HCC. METHODS AND RESULTS We examined ZBP-89 expression in five HCC cell lines and 182 HCC tissue samples by reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunofluorescence staining. Our results showed that the expression of ZBP-89 was higher in HCC than adjacent non-tumour liver, at both mRNA and protein levels. ZBP-89 was localized in the nucleus in most HCC tissue samples, but was found in the cytoplasm in 11.5% of cases. Patient survival in those tumours showing high ZBP-89 expression was better than in those with low expression. High ZBP-89 expression tended to be more common in World Health Organization (WHO) grade I than grades II-IV HCC. There was a significant association between HBV positivity and high ZBP-89 expression. Colony formation was reduced dramatically in those HCC cell lines in which ZBP-89 overexpression was demonstrated; this appeared to correlate with increased apoptosis, inferred by finding elevated levels of cleaved poly(ADP-ribose)polymerases (PARP), the probable mechanisms for which may involve increased p53 or p21 expression. CONCLUSIONS ZBP-89 has anti-tumour properties and is a potential biomarker for prognosis of HCC.
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Affiliation(s)
- Chris Z Y Zhang
- Department of Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong
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Guo C, Li D, Lin N, Huang YH, Chen ZX, Wang XZ. Establishment of a mouse model harboring the hepatitis B virus X gene and analysis of expression of apoptosis factors in liver cells of this mouse line. Shijie Huaren Xiaohua Zazhi 2011; 19:1225-1230. [DOI: 10.11569/wcjd.v19.i12.1225] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the impact of the hepatitis B virus X gene (HBVX) on the expression of apoptosis factors in liver cells.
METHODS: The eukaryotic expression vector PCDNA3.1-HBVX was injected into the caudal vein of mice to establish a mouse model expressing the HBVX gene. Mice injected with empty PCDNA3.1 vector or normal saline were used as controls. RT-PCR and Western blot were performed to detect the expression of HBVX in liver tissue 48 h after injection. Semi-quantitative RT-PCR was used to evaluate the expression of bax, bcl-2, and c-myc in liver cells of different groups of mice.
RESULTS: HBX mRNA and protein were detected in liver tissue in mice injected with the PCDNA3.1-HBVX vector, but not in controls. Compared to controls, the expression of bax, c-myc and bcl-2 was up-regulated in liver tissue in mice injected with the PCDNA3.1-HBVX vector (bax: 1.3127 ± 0.0900 vs 1.0023 ± 0.1670, 0.9094 ± 0.1081; c-myc: 1.6294 ± 0.0672 vs 1.2869 ± 0.0880, 0.9757 ± 0.0397; bcl-2: 1.5567 ± 0.1257 vs 0.6856 ± 0.1554, 0.5488 ± 0.1278, all P < 0.05).
CONCLUSION: A mouse model harboring the HBVX gene has been established successfully. Expression of the HBx gene can up-regulate the expression of Bax, c-Myc and Bcl-2 in liver cells.
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Potential targets for molecular imaging of apoptosis resistance in hepatocellular carcinoma. Biomed Imaging Interv J 2011; 7:e5. [PMID: 21655114 PMCID: PMC3107687 DOI: 10.2349/biij.7.1.e5] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2010] [Revised: 08/25/2010] [Accepted: 09/22/2010] [Indexed: 12/16/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is one of the most common cancers, which is mainly a concern in Southeast Asia. Apoptosis resistance in HCC is one of the significant factors for hepatocarcinogenesis and tumour progression. Recent advances of apoptosis resistance mechanisms in HCC could serve as potential targets for molecular imaging, which would be of considerable value to explore the molecular processes involved in HCC progression and to evaluate responses of certain anti-HCC therapies. Disruptions in the balance of anti-apoptotic and pro-apoptotic processes have been found to be involved in apoptosis resistance in HCC. Loss of response to death receptors, transformation of growth factor-β induced apoptosis, upregulation of anti-apoptotic Bcl-2 subgroup, as well as downregulation of pro-apoptotic Bax subgroup and BH3-only subgroup, are associated with apoptosis resistance in HCC. Mutation of p53 gene, dysregulation of NF-κB and survivin are also of interest because of their contribution to HCC development. In this review, the aim is to identify potential targets for molecular imaging of apoptosis resistance in HCC.
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Wang Y, Lu Y, Toh ST, Sung WK, Tan P, Chow P, Chung AYF, Jooi LLP, Lee CGL. Lethal-7 is down-regulated by the hepatitis B virus x protein and targets signal transducer and activator of transcription 3. J Hepatol 2010; 53:57-66. [PMID: 20447714 DOI: 10.1016/j.jhep.2009.12.043] [Citation(s) in RCA: 182] [Impact Index Per Article: 12.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/17/2009] [Revised: 12/28/2009] [Accepted: 12/29/2009] [Indexed: 12/12/2022]
Abstract
BACKGROUND & AIMS The pleiotropic hepatitis B virus (HBV) x protein (HBx), associated with hepatocellular carcinoma (HCC), has been implicated in the deregulation of cellular gene expression at the transcriptional level. To date, it remains unknown if HBx regulates the expression of miRNAs which play important roles in gene-regulation at the post-transcriptional and/or translational level. METHODS miRNA microarrays were employed to compare the expression of cellular miRNAs in HBx-versus control-HepG2 cells. Reverse-transcription Taqman realtime-PCR was used to examine let-7a expression in normal liver as well as paired HCC-tumor and adjacent non-tumorous liver. Let-7a miRNA was functionally characterized in cells with transiently altered let-7a expression. The direct target of let-7a was identified in silico and validated using 3'UTR-reporter assay. RESULTS HBx up-regulates 7 and down-regulates 11 miRNAs, including the let-7 family. HBx expression was found to have a significant inverse correlation with the expression of the highly-expressed members of the let-7 family in HCC patients, highlighting the clinical relevance of our observations. Further characterization of let-7a, the most highly expressed let-7 family member, revealed that it negatively regulates cellular proliferation partly through targeting signal transducer and activator of transcription 3 (STAT3). HBx-mediated down-regulation of let-7a and up-regulation of STAT3 supports cell proliferation in HBx cells. CONCLUSION This study thus represents the first demonstration of HBx's ability to deregulate cellular miRNA expression. The deregulation of the expression of the let-7 family of miRNAs by HBx may represent a potential novel pathway through which HBx acts to deregulate cell proliferation leading to hepatocarcinogenesis.
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Affiliation(s)
- Yu Wang
- Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
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Ma SH, Chen GG, Yip J, Lai PBS. Therapeutic effect of alpha-fetoprotein promoter-mediated tBid and chemotherapeutic agents on orthotopic liver tumor in mice. Gene Ther 2010; 17:905-12. [PMID: 20336154 DOI: 10.1038/gt.2010.34] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Previous studies have shown that the application of Ad/AFPtBid significantly and specifically killed hepatocellular carcinoma (HCC) cells in culture and subcutaneously implanted in mice. This study was to test the therapeutic efficacy of Ad/AFPtBid in an orthotopic hepatic tumor model. Four weeks after implantation of tumor cells into the liver, nude mice were treated with Ad/AFPtBid alone or in combination with 5-fluorouracil (5-FU). Serum alpha-fetoprotein (AFP) was measured as a marker for tumor progression. The results showed that Ad/AFPtBid significantly inhibited Hep3B tumor growth. Ad/AFPtBid and 5-FU in combination was more effective than either agent alone. Tumor tissues of Ad/AFPtBid alone or combination treatment groups showed a decrease in cells positive for proliferation cell nuclear antigen, but an increase in apoptosis. Ad/AFPtBid did not suppress the hepatic tumor formed by non-AFP-producing hepatoma SK-HEP-1 cells or colorectal adenocarcinoma DLD-1 cells. The survival rate was higher in mice treated with Ad/AFPtBid plus 5-FU than those treated with either agent alone. No acute toxic effect was observed in mice receiving Ad/AFPtBid. Collectively, Ad/AFPtBid can specifically target and effectively suppress the AFP-producing orthotopic liver tumor in mice without obvious toxicity, indicating that it is a promising tool in combination with chemotherapeutic agents for treatment of AFP-producing HCC.
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Affiliation(s)
- S-H Ma
- Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong
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15
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Gramantieri L, Fornari F, Ferracin M, Veronese A, Sabbioni S, Calin GA, Grazi GL, Croce CM, Bolondi L, Negrini M. MicroRNA-221 targets Bmf in hepatocellular carcinoma and correlates with tumor multifocality. Clin Cancer Res 2009; 15:5073-81. [PMID: 19671867 DOI: 10.1158/1078-0432.ccr-09-0092] [Citation(s) in RCA: 249] [Impact Index Per Article: 15.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
UNLABELLED Deregulated cell proliferation and apoptosis play a major role in hepatocellular carcinoma (HCC). MicroRNAs participate in the modulation of key molecules linked to hepatocarcinogenesis. PURPOSE This study aims to investigate the role of miR-221 in the modulation of Bmf, a proapoptotic BH3-only protein, and to characterize miR-221 contribution to hepatocarcinogenesis through modulation of apoptosis. EXPERIMENTAL DESIGN Transfection of miR-221 and anti-miR-221 in HCC-derived cell lines and luciferase reporter assay were used to assess Bmf as a target of miR-221. Modulation of miR-221 and Bmf expression contributed to characterize their role in anoikis. Primary HCC tissues were analyzed to assess the clinical relevance of in vitro findings. RESULTS Enforced miR-221 expression caused Bmf down-regulation, whereas anti-miR-221 induced its up-regulation. A luciferase reporter assay confirmed Bmf as a target of miR-221. Following matrix detachment, miR-221 silencing led to increased apoptotic cell death. The analysis of HCC tissues revealed an inverse correlation between miR-221 and Bmf expression and a direct correlation between Bmf and activated caspase-3, as a marker of apoptosis. High miR-221 levels were associated with tumor multifocality and reduced time to recurrence after surgery. CONCLUSIONS Our results indicate that miR-221, by targeting Bmf, inhibits apoptosis. Moreover, in HCC, miR-221 overexpression is associated with a more aggressive phenotype. These findings, together with the previously reported modulation of CDKN1B/p27 and CDKN1C/p57, show that miR-221 simultaneously affects multiple pro-oncogenic pathways and suggest miR-221 as a potential target for nonconventional treatment against HCC.
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MESH Headings
- Adaptor Proteins, Signal Transducing/genetics
- Aged
- Aged, 80 and over
- Apoptosis/drug effects
- Apoptosis/genetics
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/mortality
- Carcinoma, Hepatocellular/pathology
- Female
- Gene Expression Regulation, Neoplastic
- Gene Targeting
- Humans
- Liver Neoplasms/genetics
- Liver Neoplasms/mortality
- Liver Neoplasms/pathology
- Male
- MicroRNAs/antagonists & inhibitors
- MicroRNAs/physiology
- Middle Aged
- Neoplasm Recurrence, Local
- Neoplasms, Multiple Primary/genetics
- Neoplasms, Multiple Primary/mortality
- Neoplasms, Multiple Primary/pathology
- RNA, Small Interfering/pharmacology
- Tumor Cells, Cultured
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Affiliation(s)
- Laura Gramantieri
- Dipartimento di Medicina Interna e Gastroenterologia e Centro di Ricerca Biomedica Applicata, Università di Bologna, Bologna, Italy.
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16
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Gramantieri L, Fornari F, Callegari E, Sabbioni S, Lanza G, Croce CM, Bolondi L, Negrini M. MicroRNA involvement in hepatocellular carcinoma. J Cell Mol Med 2009; 12:2189-204. [PMID: 19120703 PMCID: PMC4514099 DOI: 10.1111/j.1582-4934.2008.00533.x] [Citation(s) in RCA: 211] [Impact Index Per Article: 13.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. Curative options for HCC are limited and exclusively available for patients carrying an early stage HCC. In advanced stages, traditional chemotherapy proved to be only marginally effective or even toxic. Thus, the identification of new treatment options is needed. New targets for non-conventional treatment will necessarily take advantage of progresses on the molecular pathogenesis of HCC. MicroRNAs (miRNAs) are a group of tiny RNAs with a fundamental role in the regulation of gene expression. Aberrant expression of several miRNAs was found to be involved in human hepatocarcinogenesis. miRNA expression signatures were correlated with bio-pathological and clinical features of HCC. In some cases, aberrantly expressed miRNAs could be linked to cancer-associated pathways, indicating a direct role in liver tumourigenesis. For example, up-regulation of mir-221 and mir-21 could promote cell cycle progression, reduce cell death and favour angiogenesis and invasion. These findings suggest that miRNAs could become novel molecular targets for HCC treatment. The demonstration of in vivo efficacy and safety of anti-miRNA compounds has opened the way to their use in clinical trials.
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Affiliation(s)
- Laura Gramantieri
- Department of Internal Medicine and Gastroenterology, University of Bologna, Bologna, Italy
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17
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Abstract
Hepatocellular carcinoma (HCC) is a major health problem, being the sixth most common cancer world-wide. Dysregulation of the balance between proliferation and cell death represents a pro-tumorigenic principle in human hepatocarcinogenesis. This review updates the recent relevant contributions reporting molecular alterations for HCC that induce an imbalance in the regulation of apoptosis. Alterations in the expression and/or activation of p53 are frequent in HCC cells, which confer on them resistance to chemotherapeutic drugs. Many HCCs are also insensitive to apoptosis induced either by death receptor ligands, such as FasL or TRAIL, or by transforming growth factor-beta (TGF-β). Although the expression of some pro-apoptotic genes is decreased, the balance between death and survival is dysregulated in HCC mainly due to overactivation of anti-apoptotic pathways. Indeed, some molecules involved in counteracting apoptosis, such as Bcl-XL, Mcl-1, c-IAP1, XIAP or survivin are over-expressed in HCC cells. Furthermore, some growth factors that mediate cell survival are up-regulated in HCC, as well as the molecules involved in the machinery responsible for cleavage of their pro-forms to an active peptide. The expression and/or activation of the JAK/STAT, PI3K/AKT and RAS/ERKs pathways are enhanced in many HCC cells, conferring on them resistance to apoptotic stimuli. Finally, recent evidence indicates that inflammatory processes, as well as the epithelial-mesenchymal transitions that occur in HCC cells to facilitate their dissemination, are related to cell survival. Therefore, therapeutic strategies to selectively inhibit anti-apoptotic signals in liver tumor cells have the potential to provide powerful tools to treat HCC.
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18
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Putative roles of hepatitis B x antigen in the pathogenesis of chronic liver disease. Cancer Lett 2009; 286:69-79. [PMID: 19201080 DOI: 10.1016/j.canlet.2008.12.010] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2008] [Revised: 11/18/2008] [Accepted: 12/02/2008] [Indexed: 12/18/2022]
Abstract
Under most circumstances, hepatitis B virus (HBV) is noncytopathic. However, hepatocellular regeneration that accompanies each bout of hepatitis appears to be associated with increased integration of HBV DNA fragments expressing the virus encoded hepatitis B x antigen (HBxAg). Intrahepatic HBxAg staining correlates with the intensity and progression of chronic liver disease (CLD), and additional work has shown that HBxAg blocks immune mediated killing by Fas and by tumor necrosis factor alpha (TNFalpha). This is not only associated with the blockage of caspase activities by HBxAg, but also by the constitutive stimulation of hepatoprotective pathways, such as nuclear factor kappa B (NF-kappaB), phosphoinositol 3-kinase (PI3K), and beta-catenin (beta-catenin). HBxAg also appears to promote fibrogenesis, by stimulating the production of fibronectin. HBxAg also stimulates the production and activity of transforming growth factor beta1 (TGFbeta1) by several mechanisms, thereby promoting the profibrogenic and tumorigenic properties of this important cytokine. In addition, HBxAg appears to remodel the extracellular matrix (ECM) by altering the expression of several matrix metalloproteinases (MMPs), which may promote tumor metastasis. Hence, HBxAg appears to promote chronic infection by preventing immune mediated apoptosis of infected hepatocytes, by promoting the establishment and persistence of fibrosis and cirrhosis preceding the development of HCC, and by promoting the remodeling of EMC during tumor progression.
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19
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Park NH, Chung YH. [Molecular mechanisms of hepatitis B virus-associated hepatocellular carcinoma]. THE KOREAN JOURNAL OF HEPATOLOGY 2008; 13:320-40. [PMID: 17898549 DOI: 10.3350/kjhep.2007.13.3.320] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Hepatocellular carcinoma (HCC) is one of the most common malignant diseases in the world. The hepatitis B virus (HBV) replicates non-cytopathically in hepatocytes, and most of the liver injury associated with this infection reflects the immune response. Epidemiological studies have clearly demonstrated that a chronic HBV infection is a major etiological factor in the development of HCC. The pathogenesis of HBV-associated HCC has been studied extensively, and the molecular changes during the malignant transformation have been identified. The main carcinogenic mechanism of HBV-associated HCC is related to the long term-inflammatory changes caused by a chronic hepatitis B infection, which might involve the integration of the HBV. Integration of the HBV DNA into the host genome occurs at the early steps of clonal tumorous expansion. The hepatitis B x protein (HBx) is a multifunctional regulatory protein that communicates directly or indirectly with a variety of host targets, and mediates many opposing cellular functions, including its function in cell cycle regulation, transcriptional regulation, signaling, encoding of the cytoskeleton and cell adhesion molecules, as well as oncogenes and tumor suppressor genes. Continued study of the mechanisms of hepatocarcinogenesis will refine our current understanding of the molecular and cellular basis for neoplastic transformations in the liver. This review summarizes the current knowledge of the mechanisms involved in HBV-associated hepatocarcinogenesis.
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Affiliation(s)
- Neung Hwa Park
- Department of Internal Medicine, University of Ulsan College of Medicine, Biomedical Research Center, Ulsan University Hospital, Ulsan, Korea
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20
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Lu YW, Ren YD, Bai J, Chen WN. The spliced variant of hepatitis B virus protein, HBSP, interacts with Bcl-2/Bcl-xlin vitroand induces apoptosis in HepG2 cells. IUBMB Life 2008; 60:700-2. [DOI: 10.1002/iub.108] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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21
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Park NH, Song IH, Chung YH. Molecular Pathogenesis of Hepatitis-B-virus-associated Hepatocellular Carcinoma. Gut Liver 2007; 1:101-17. [PMID: 20485626 DOI: 10.5009/gnl.2007.1.2.101] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/03/2007] [Accepted: 11/18/2007] [Indexed: 12/18/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is one of the most frequent and malignant diseases worldwide. Epidemiological studies have clearly demonstrated that chronic hepatitis B virus (HBV) infection is a major etiological factor in the development of HCC. The pathogenesis of HBV-associated HCC has been studied extensively, and the molecular changes associated with malignant transformation have been identified. The predominant carcinogenic mechanisms of HBV-associated HCC are chronic inflammation and the effects of cytokines in the development of fibrosis and liver cell proliferation. An important role is also played by the integration of HBV DNA into host cellular DNA, which disrupts or promotes the expression of cellular genes that are important in cell growth and differentiation. Especially, HBx protein is a transactivating protein that promotes cell growth, survival, and the development of HCC. Continued investigation of the mechanisms underlying hepatocarcinogenesis will refine our current understanding of the molecular and cellular basis for neoplastic transformation in the liver. Prevention of HBV infections and effective treatments for chronic hepatitis B are still needed for the global control of HBV-associated HCC. This review summarizes the current knowledge on the mechanisms involved in HBV-associated hepatocarcinogenesis.
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Affiliation(s)
- Neung Hwa Park
- Division of Gastroenterology, Department of Internal Medicine, University of Ulsan College of Medicine, Ulsan University Hospital, Ulsan, Korea
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22
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The hepatitis B x antigen effector, URG7, blocks tumour necrosis factor α-mediated apoptosis by activation of phosphoinositol 3-kinase and β-catenin. J Gen Virol 2007; 88:3275-3285. [DOI: 10.1099/vir.0.83214-0] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
Hepatitis B x antigen (HBxAg) contributes significantly to the pathogenesis of chronic infection and development of hepatocellular carcinoma. To discern some of its operative pathways, HepG2 cells were stably transduced with HBx or the bacterial chloramphenicol acetyltransferase (CAT) gene. Differential gene expression has previously revealed an upregulated gene, clone 7 (URG7), that conferred resistance to anti-Fas killing on HepG2X cells. Given that tumour necrosis factor alpha (TNFα) is also an important mediator of chronic hepatitis, and partially shares signalling with Fas, experiments were designed to test whether URG7 blocks TNFα killing of HepG2X cells. HepG2X cells expressing URG7 and HepG2 cells overexpressing URG7 in the absence of HBxAg were resistant to TNFα killing compared with HepG2CAT cells. URG7 small interfering RNA restored the sensitivity of HepG2X cells to TNFα killing. Killing was associated with the activation of caspases 3 and 8, suggesting that URG7 blocked these caspases. This resistance was also associated with activation of phosphoinositol 3-kinase/Akt. Given that Akt and HBxAg also activate β-catenin, experiments were designed to determine whether URG7 blocked apoptosis via activation of β-catenin. Both HBxAg and URG7 activated fragments of the β-catenin promoter, and also promoted expression of β-catenin target genes. Hence, URG7 inhibits TNFα-mediated killing by blocking one or more caspases in the apoptotic pathway and by activating phosphoinositol 3-kinase and β-catenin, thereby overriding the apoptotic signalling of TNFα. This suggests that URG7 helps to protect virus-infected hepatocytes during chronic hepatitis B virus infection.
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23
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Lu YW, Tan TL, Zhang J, Chen WN. Cellular apoptosis induced by replication of hepatitis B virus: possible link between viral genotype and clinical outcome. Virol J 2007; 4:117. [PMID: 17973991 PMCID: PMC2174443 DOI: 10.1186/1743-422x-4-117] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2007] [Accepted: 10/31/2007] [Indexed: 12/15/2022] Open
Abstract
HBV remains one of the major pathogens of liver diseases but the outcomes as inflammation, cirrhosis and cancer of the liver are greatly related to different viral genotypes. The aim of this study was to assess the pro-apoptotic effect of HBSP from three HBV genotypes on liver derived cells. HepG2 cells were applied in our system and transfected by HBV genotype A, B, and C. Cells were observed under phase contrast microscope, stained by apoptosis marker and analyzed by flow cytometre. HBSP expression was detected by western blot assay. BH3 sequences were aligned and analyzed by Vector NTI. HBV genotypes A, B, and C transfected cells displayed evidence of cell death which was further proved as apoptosis. Natural expression of a pro-apoptotic protein HBSP was detected during genomes transfection. The different apoptotic effects were correlated to the HBSP expression from each genome. Alignment and analysis of the BH3 domains from the three genomes revealed slight variance which might also contribute to the result. Our results suggested that variant HBSP expression and BH3 sequence of HBV genotypes may be involved in differential apoptotic effect in transfected cells. Detailed analysis of the role of HBV genotypes in cellular apoptotic process should provide molecular information on the reported clinical outcome of infection by different HBV genotypes.
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Affiliation(s)
- Yi Wei Lu
- School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, 637459, Singapore.
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24
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Miao J, Chen GG, Yun JP, Chun SY, Zheng ZZ, Ho RLK, Chak ECW, Xia NS, Lai PBS. Identification and characterization of BH3 domain protein Bim and its isoforms in human hepatocellular carcinomas. Apoptosis 2007; 12:1691-701. [PMID: 17503221 DOI: 10.1007/s10495-007-0093-5] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
BH3-only protein Bim is a critical regulator of apoptosis and plays an essential role in mammalian development, but the characterization of Bim and its isoforms in hepatocellular carcinoma (HCC) has not been studied before. Here we investigated the expression, distribution, regulation and role of Bim isoforms in HCC cells. Fifteen Bim isoforms were identified in HCC, with six newly identified isoforms and two newly identified exons. Among of them, Bim EL, L, S, a1, a2, a3, b2, b4 and b6 are abundant isoforms according to their mRNA levels. However only Bim EL, L and S proteins could be clearly detected. Bim mRNA and protein were strongly expressed in HCC tissues compared to relevant non-tumorous regions, but the ratio of variant isoforms showed no difference between tumorous and non-tumorous tissues. Bim isoforms were differentially regulated after chemotherapeutic drug 5-Fluorouracil (5-FU) treatment. Interestingly, Bim EL, L and S, the isoforms known to induce apoptosis strongly, are the least inducible isoforms at their mRNA levels when exposed to the stress, suggesting that post-transcriptional rather than transcriptional, modulations may play a role to enhance their functions. Finally, overexpression of Bim EL, L, S and all alpha isoforms induced apoptosis in HCC cells, while overexpression of Bim beta isoforms showed no effects on cell survival after 5-FU treatment. In conclusion, Bim alpha isoforms appears to have a role in the regulation of apoptosis in HCC cells, which may contribute to not only the growth of tumor cells but also the sensitivity of HCC cells to chemotherapy.
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Affiliation(s)
- Ji Miao
- Department of Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong
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25
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Abstract
BACKGROUND/AIMS Dysregulation of the balance between proliferation and cell death represents a protumorigenic principle in human hepatocarcinogenesis. This article aims to provide a review of the current findings about how physiological hepatocyte apoptosis is regulated and whether or not its dysregulation might contribute to the progression towards a hepatocellular carcinoma (HCC) process. RESULTS Although some physiological proapoptotic molecules are downregulated or inactivated in HCC, such as Fas, p53, Bax or Bid, dysregulation of the balance between death and survival is mainly due to overactivation of antiapoptotic signals. Thus, some growth factors that mediate cell survival are upregulated in HCC, as well as the molecules involved in the machinery responsible for cleavage of their proforms to an active peptide. The expression of the pten gene is reduced or absent in almost half the HCCs and the Spred family of Ras/ERK inhibitors is also dysregulated in HCC, which consequently lead to the overactivation of relevant survival kinases: AKT and ERKs. Alterations in the expression and/or activity of molecules involved in counteracting apoptosis, such as NF-kappaB, Bcl-X(L), Mcl-1 or c-IAP1, have also been observed in HCC. CONCLUSIONS Therefore, therapeutic strategies to inhibit selectively antiapoptotic signals in tumour cells have the potential to provide powerful tools to treat liver cancer.
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Affiliation(s)
- Isabel Fabregat
- Institut de Investigació Biomèdica de Bellvitge, Institut de Recerca Oncològica, L'Hospitalet, Barcelona, Spain.
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Miao J, Chen GG, Chun SY, Yun JP, Chak ECW, Ho RLK, Lai PBS. Adenovirus-mediated tBid overexpression results in therapeutic effects on p53-resistant hepatocellular carcinoma. Int J Cancer 2006; 119:1985-93. [PMID: 16708390 DOI: 10.1002/ijc.22040] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with a very high mortality. Because the success of the conventional therapies is limited, gene therapy may represent an alternative for HCC management. Our earlier study has shown that Bid plays a role in the development of HCC. The aim of our study is to evaluate the possibility of using truncated Bid (tBid) as a novel therapy for HCC treatment. Two HCC cell lines, Hep3B and PLC/PRF/5, were used in the experiment. Hep3B was a p53-resistant while PLC/PRF/5 a p53-sensitive. A recombinant adenovirus-Ad/AFPtBid, which contained a tBid gene driven by an alpha-fetoprotein (AFP) promoter, was constructed. Both Hep3B and PLC/PRF/5 cells infected with Ad/AFPtBid showed a significant decrease in cell viability. The decrease in cell viability by Ad/AFPtBid resulted from apoptosis of HCC cells, evident by enhanced activity of caspases and increased release of cytochrome c. In vivo experiment was performed by the intratumor injection of Ad/AFPtBid in nude mice inoculated with Hep3B. Ad/AFPtBid injection significantly inhibited tumor growth, and tumor tissues showed a marked increase in TUNEL-positive cells. Our experiment also demonstrated that Ad/AFPtBid only targeted AFP-producing cells but not those non-AFP producing cells. In conclusion, these results indicate that the introduction of Ad/AFPtBid can not only significantly but specifically kill HCC cells that produce AFP. The cell death induced by Ad/AFPtBid in HCC cells is via an apoptotic pathway that can be independent of p53 status.
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Affiliation(s)
- Ji Miao
- Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong
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Lu YW, Tan TL, Chan V, Chen WN. The HBSP gene is expressed during HBV replication, and its coded BH3-containing spliced viral protein induces apoptosis in HepG2 cells. Biochem Biophys Res Commun 2006; 351:64-70. [PMID: 17049490 DOI: 10.1016/j.bbrc.2006.10.002] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2006] [Accepted: 10/01/2006] [Indexed: 01/14/2023]
Abstract
The mechanisms of liver injury in hepatitis B virus (HBV) infection are defined to be due not to the direct cytopathic effects of viruses, but to the host immune response to viral proteins expressed by infected hepatocytes. We showed here that transfection of mammalian cells with a replicative HBV genome causes extensive cytopathic effects, leading to the death of infected cells. While either necrosis or apoptosis or both may contribute to the death of infected cells, results from flow cytometry suggest that apoptosis plays a major role in HBV-induced cell death. Data mining of the four HBV protein sequences reveals the presence of a Bcl-2 homology domain 3 (BH3) in HBSP, a spliced viral protein previously shown to be able to induce apoptosis and associated with HBV pathogenesis. HBSP is expressed at early stage of our cell-based HBV replication. When transfected into HepG2 cells, HBSP causes apoptosis in a caspase dependent manner. Taken together, our results suggested a direct involvement of HBV viral proteins in cellular apoptosis, which may contribute to liver pathogenesis.
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Affiliation(s)
- Yi Wei Lu
- School of Biological Sciences, College of Engineering, Nanyang Technological University, Singapore 637722, Singapore
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28
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N/A, 林 纳. N/A. Shijie Huaren Xiaohua Zazhi 2006; 14:2579-2585. [DOI: 10.11569/wcjd.v14.i26.2579] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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Park NH, Song IH, Chung YH. Chronic hepatitis B in hepatocarcinogenesis. Postgrad Med J 2006; 82:507-15. [PMID: 16891440 PMCID: PMC2585715 DOI: 10.1136/pgmj.2006.047431] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2006] [Accepted: 04/07/2006] [Indexed: 01/18/2023]
Abstract
Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world, and has a wide geographical variation. Eighty per cent of HCC is attributed to hepatitis B virus (HBV). The predominant carcinogenic mechanism of HBV associated HCC is through the process of liver cirrhosis, but direct oncogenic effects of HBV may also contribute. Prevention of HBV infections as well as effective treatment of chronic hepatitis B is still needed for the global control of HBV associated HCC. Continued investigation of the mechanisms of hepatocarcinogenesis will refine our current understanding of the molecular and cellular basis for neoplastic transformation in the liver.
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Affiliation(s)
- N H Park
- Division of Gastroenterology, Department of Internal Medicine, University of Ulsan, College of Medicine, Asan Medical Centre, Seoul, Korea
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30
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Chen GG, Li MY, Ho RLK, Chak ECW, Lau WY, Lai PBS. Identification of hepatitis B virus X gene mutation in Hong Kong patients with hepatocellular carcinoma. J Clin Virol 2005; 34:7-12. [PMID: 16087118 DOI: 10.1016/j.jcv.2005.01.006] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2004] [Revised: 12/01/2004] [Accepted: 01/01/2005] [Indexed: 01/03/2023]
Abstract
BACKGROUND Chronic infection by hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) in man. The viral transactivator HBV X (HBx) gene plays a critical role in the molecular pathogenesis of HBV-related HCC. OBJECTIVES The aim of this study was to investigate whether there were particular HBx mutations associated with the Chinese Hong Kong patients with HCC. STUDY DESIGN We have examined HBx in 113 tumor tissue samples from patients with HCC and 48 serum samples from the same group. In addition, we also examined the expression of HBx protein and the index of apoptotic cell death in tumor tissues of HCC. The entire coding region of HBx gene from the sample was sequenced and aligned with the published HBx gene sequence. RESULTS AND CONCLUSIONS We have identified total 54 different types of mutations in HBx gene. HBx mutations occurred in a very high percentage of samples tested. Mutation of HBx was found in 95.2% and 95.3% of the tumor tissue and serum samples, respectively. Most of samples contained more than one type of the mutation. Relative risk analysis indicated that the mutations in 12 sites of tissue HBx and nine sites of serum HBx were highly associated with HCC, suggesting a potential role of these mutants in carcinogenesis. An insert mutation at position 204: Insert 204AGGCCC, was always found to co-exist with point mutations at 260 (G-->A) and 264 (G/C/T-->A). Furthermore, this particular pattern of HBx mutation was most frequently detected. Immunochemical staining of HBx protein revealed that the nuclear localization of HBx protein in hepatocytes of tumor tissues was highly associated with this particular pattern of HBx mutation. In conclusion, HBx mutation occurs frequently in HCC samples tested and a sample usually has multiple types of mutations. A special pattern of insert at 204 and point mutations at 260 and 264 was identified, and it appears to be associated with the nuclear localization of HBx protein. The development of multiple types of mutations in a given sample may contribute to the process of multiple steps in hepatocarcinogenesis.
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Affiliation(s)
- G G Chen
- Department of Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, Room 306 Cancer Center, Shatin, New Territories, Hong Kong.
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31
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Lu YW, Chen WN. Human hepatitis B virus X protein induces apoptosis in HepG2 cells: Role of BH3 domain. Biochem Biophys Res Commun 2005; 338:1551-6. [PMID: 16274670 DOI: 10.1016/j.bbrc.2005.10.117] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2005] [Accepted: 10/19/2005] [Indexed: 11/25/2022]
Abstract
The smallest protein of hepatitis B virus, HBX, has been implicated in the development of liver diseases by interfering with normal cellular processes. Its role in cell proliferation has been unclear as both pro-apoptotic and anti-apoptotic activities have been reported. We showed molecular evidence that HBX induced apoptosis in HepG2 cells. A Bcl-2 Homology Domain 3 was identified in HBX, which interacted with anti-apoptotic but not pro-apoptotic members of the Bcl-2 family of proteins. HBX induced apoptosis when transfected into HepG2 cells, as demonstrated by both flow cytometry and caspase-3 activity. However, HBX protein may not be stable in apoptotic cells triggered by its own expression as only its mRNA or the fusion protein with the glutathione-S-transferase was detected in transfected cells. Our results suggested that HBX behaved as a pro-apoptotic protein and was able to induce apoptosis.
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Affiliation(s)
- Yi Wei Lu
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive 05N-10, Singapore 637551, Singapore
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Zhang X, Dong N, Yin L, Cai N, Ma H, You J, Zhang H, Wang H, He R, Ye L. Hepatitis B virus X protein upregulates survivin expression in hepatoma tissues. J Med Virol 2005; 77:374-81. [PMID: 16173017 DOI: 10.1002/jmv.20466] [Citation(s) in RCA: 70] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
The hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). The relationship was examined between HBV antigens and IAP (inhibitor of apoptosis) family in development of HCC. The expression levels of HBV antigens (HBsAg, HBcAg, and HBxAg) and members of the IAP family (survivin, XIAP, cIAP-1, and cIAP-2) were detected immunohistochemically in tissues from 34 cases of HCC and 30 cases of liver cirrhosis. The positive rate of survivin was higher than these three molecules in all three tissue types (P < 0.05). The positive rates of HBxAg and survivin were high in HCC (76.5% and 88.2%), paratumor (85.3% and 91.2%), and liver cirrhosis (100% and 93.3%) tissues, with no significant differences between the survivin- and HBxAg-positive rates (each P > 0.05). To examine the effect of HBx on survivin expression, plasmid pCMV-X (encoding the HBx gene) was transfected transiently with or without plasmid pcDNA3-sur (encoding the survivin gene) into H7402 hepatoma cells and L-O2 human normal liver cells. Cells over-expressing HBx alone showed increased apoptosis along with a dose-dependent increase in survivin levels. However, co-expression of survivin inhibited the HBx-induced apoptosis. To examine the effect of HBx on survivin in hepatoma cells without apoptosis, plasmid pCMV-X was transfected stably into human hepatoma H7402 cells and L-O2 cells. These H7402-X and L-O2-X cells showed high-level expression of both HBx and survivin, but did not show apoptosis. The addition of pSilencer 3.0-X, an RNAi vector targeting the HBx gene, reduced the expression levels of survivin protein in H7402-X cells. Collectively, these data demonstrate that HBx upregulates survivin expression in hepatoma tissues, suggesting that HBx and survivin may both be involved in carcinogenesis of HCC.
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Affiliation(s)
- Xiaodong Zhang
- Department of Cancer Research, Institute for Molecular Biology, Tianjin Key Laboratory of Microbial Functional Genomics, College of Life Sciences, Nankai University, Tianjin, P.R. China.
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Zhang SJ, Chen HY, Chen ZX, Wang XZ. Possible mechanism for hepatitis B virus X gene to induce apoptosis of hepatocytes. World J Gastroenterol 2005; 11:4351-6. [PMID: 16038033 PMCID: PMC4434661 DOI: 10.3748/wjg.v11.i28.4351] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the possible mechanism for HBV X gene to induce apoptosis of hepatocyte HL-7702 cells.
METHODS: HBV X gene eukaryon expression vector pcDNA3-X was established and transfected into HL-7702 cells by lipid-mediated transfection, including transient and stable transfection. Positive clones were screened by incubating in the selective medium with 600 mg/mL G418 and named HL-7702/HBV-encoded X protein (HBx) cells. The expressions of Fas/FasL, Bax/Bcl-2, and c-myc mRNA were measured by semi-quantitative RT-PCR in HL-7702/HBx and control group, respectively.
RESULTS: RT-PCR analysis confirmed that HBV X gene was transfected into HL-7702 cells successfully. By semi-quantitative RT-PCR analysis, Bax and c-myc mRNA levels in HL-7702/HBx cells of transient transfection were significantly higher than those in control, FasL and c-myc mRNA levels in HL-7702/HBx cells of stable transfection were significantly higher than those in control, whereas the Bcl-2 mRNA levels in HL-7702/HBx cells of transient and stable transfection were significantly lower than those in control.
CONCLUSION: HBV X gene may promote the apoptosis of hepatocytes by regulating the expressions of Fas/FasL, Bax/Bcl-2, and c-myc gene in a dose-dependent manner.
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Affiliation(s)
- Sheng-Jun Zhang
- Department of Gastroenterology, Union Hospital of Fujian Medical University, Fuzhou 350001, Fujian Province, China
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Abstract
AIM: To investigate the effect of hepatitis B virus (HBV) X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells.
METHODS: The HBV X gene eukaryon expression vector pcDNA3-X was transiently transfected into HepG2 cells by lipid-media transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3 were used as controls. Expression of HBx in HepG2 was identified by RT-PCR. MTT and TUNEL were employed to measure proliferation and apoptosis of cells in three groups. Semi-quantified RT-PCR was used to evaluate the expression levels of Fas/FasL, Bax/Bcl-xL, and c-myc in each group.
RESULTS: HBV X gene was transfected into HepG2 cells successfully. RT-PCR showed that HBx was only expressed in HepG2/pcDNA3-X cells, but not expressed in HepG2 and HepG2/pcDNA3 cells. Analyzed by MTT, cell proliferation capacity was obviously lower in HepG2/pcDNA3-X cells (0.08910±0.003164) than in HepG2 (0.14410±0.004927) and HepG2/pcDNA3 cells (0.12150±0.007159) (P<0.05 and P<0.01). Analyzed by TUNEL, cell apoptosis was much more in HepG2/pcDNA3-X cells (980/2 000) than HepG2 (420/2 000), HepG2/pcDNA3 cells (520/2 000) (P<0.05 and P<0.01). Evaluated by semi-quantified RT-PCR, the expression level of Fas/FasL was significantly higher in HepG2 cells transfected with HBx than in HepG2 and HepG2/pcDNA3cells (P<0.05 and P<0.01). Bax/Bcl-xL expression level was also elevated in HepG2/pcDNA3-X cells (P<0.05 and P<0.01). Expression of c-myc was markedly higher in HepG2/pcDNA3-X cells than in HepG2 and HepG2/pcDNA3 cells (P<0.05 andP<0.01).
CONCLUSION: HBV X gene can impair cell proliferation capacity, improve cell apoptosis, and upregulate expression of apoptosis factors. The intervention of HBV X gene on the expression of apoptosis factors may be a possible mechanism responsible for the change in cell apoptosis and proliferation.
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Affiliation(s)
- Na Lin
- Department of Gastroenterology, Affiliated Union Hospital, Fujian Medical University, Fuzhou 350001, Fujian Province, China
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Miao J, Chen GG, Chun SY, Lai PPS. Hepatitis B virus X protein induces apoptosis in hepatoma cells through inhibiting Bcl-xL expression. Cancer Lett 2005; 236:115-24. [PMID: 15990224 DOI: 10.1016/j.canlet.2005.05.014] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2005] [Revised: 05/04/2005] [Accepted: 05/06/2005] [Indexed: 02/06/2023]
Abstract
The X protein of hepatitis B virus (HBx) exhibits numerous activities affecting gene transcription, intracellular signal transduction, cell proliferation and apoptosis. Recent studies showed that HBx induced apoptosis by causing loss of mitochondrial membrane potential, suggesting that HBx-mediated apoptosis is mitochondria-dependent. However, the molecular mechanism of the gene in this pathway is still far from understood. In this study, we demonstrated that introduction of HBx into a hepatocellular carcinoma cell line, Hep3B, caused apoptosis and sensitized the cell to TNFalpha-induced cell killing. Over-expression of Bcl-xL, an anti-apoptotic Bcl-2 family protein, prevented cell death dragged by HBx. Importantly, expression of HBx in Hep3B cells reduced Bcl-xL mRNA and protein levels, but did not regulate other Bcl-2 family members. Although, HBx itself did not affect intracellular distribution of cytochrome c, an enhanced release of cytochrome c from mitochondria was observed when TNFalpha was applied. Thus, the introduction of HBx into Hep3B cells induces apoptosis and sensitizes Hep3B cells to TNFalpha-mediated cell killing, and these processes may accomplish through inhibiting Bcl-xL expression and subsequently promoting cytochrome c release from mitochondria.
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Affiliation(s)
- Ji Miao
- Department of Surgery, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong SAR
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Kountouras J, Zavos C, Chatzopoulos D. Apoptotic and anti-angiogenic strategies in liver and gastrointestinal malignancies. J Surg Oncol 2005; 90:249-59. [PMID: 15906369 DOI: 10.1002/jso.20254] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Inappropriate suppression of apoptosis is strongly implicated in tumorigenesis. Tumor development is heralded by the mutation of tumor suppressor genes and overexpression of anti-apoptotic genes permitting cell survival. Thus, inducing the apoptotic process in various ways can be applied to cancer management. Besides, angiogenesis is a crucial process for tumor growth and metastasis. New strategies targeting fundamental play-markers of the angiogenic process are currently under investigation.
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Affiliation(s)
- Jannis Kountouras
- Department of Medicine, Second Medical Clinic, Aristotle University of Thessaloniki, Ippokration Hospital, Thessaloniki, Greece.
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Chen GG, Lai PBS, Ho RLK, Chan PKS, Xu H, Wong J, Lau WY. Reduction of double-stranded RNA-activated protein kinase in hepatocellular carcinoma associated with hepatitis B virus. J Med Virol 2004; 73:187-94. [PMID: 15122791 DOI: 10.1002/jmv.20074] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) in Asia. Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an interferon-induced, serine/threonine protein kinase. Recent studies have suggested that PKR is involved in the pathogenesis of HCC with hepatitis virus C infection by inhibiting viral and cellular proteins related to cell growth and proliferation. In the present study, PKR was examined in both tumor and non-tumor tissues from HCC livers infected with HBV. The expression of PKR was determined by TaqMan real-time PCR and immunohistochemical methods. The level of PKR was also analyzed in relation to pathological changes observed in HCC. The result showed that PKR was reduced in tumor tissues of HCC from HBV carriers with low serum viral load (<0.7 x 10(6) copies/ml) compared to those with higher serum viral load. However, the overall PKR level was much lower in tumor tissues than that in non-tumor tissues, irrespective of HBV carrier status or serum viral load. PKR level tended to be lower in HCC samples with alpha-fetoprotein (AFP) more than 500 ng/ml (mean: 4024.2 ng/ml) than those with AFP less than 500 ng/ml (mean: 50.6 ng/ml). There was no significant difference in the expression of PKR between tumor tissues with well differentiation and those with poor or moderate differentiation. In conclusion, the level of PKR was reduced in HCC tumor tissues, suggesting a possible role of PKR in promoting the growth of tumor. HBV may participate in altering the level of PKR, but factors other than HBV should play a more determining role in the regulation of PKR in HCC. The association between PKR and AFP levels may offer an alternative tumor marker for HCC.
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MESH Headings
- Adult
- Aged
- Biomarkers, Tumor
- Biopsy
- Carcinoma, Hepatocellular/enzymology
- Carcinoma, Hepatocellular/metabolism
- Carcinoma, Hepatocellular/pathology
- Carcinoma, Hepatocellular/virology
- DNA, Viral/blood
- DNA, Viral/isolation & purification
- Female
- Gene Expression
- Hepatitis B virus/isolation & purification
- Hepatitis B, Chronic/complications
- Hepatitis B, Chronic/virology
- Humans
- Liver/enzymology
- Liver/pathology
- Liver Neoplasms/enzymology
- Liver Neoplasms/metabolism
- Liver Neoplasms/pathology
- Male
- Middle Aged
- RNA, Messenger/analysis
- RNA, Messenger/isolation & purification
- Viral Load
- alpha-Fetoproteins/analysis
- eIF-2 Kinase/biosynthesis
- eIF-2 Kinase/genetics
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Affiliation(s)
- George G Chen
- Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, N.T. Hong Kong.
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Yokoo H, Kondo T, Fujii K, Yamada T, Todo S, Hirohashi S. Proteomic signature corresponding to alpha fetoprotein expression in liver cancer cells. Hepatology 2004; 40:609-17. [PMID: 15349899 DOI: 10.1002/hep.20372] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Alpha fetoprotein (AFP) has been implicated in the development of hepatocellular carcinoma and is considered to be a diagnostic and prognostic tumor marker. Because elevated expression of AFP is associated with many characteristics of hepatocellular carcinoma tissues, we hypothesized that multiple proteins may function in a coordinated manner with AFP. To identify such proteins, we performed global protein expression analysis, namely a proteomic study. The protein expression profiles of 9 AFP-producing liver cancer cell lines (JHH-5, HuH-1, PLC/PRL/5, Hep3B, HT-17, JHH-7, HuH-7, HepG2, Li-7) and 7 nonproducing liver cancer cell lines (HLE, JHH-6, Sk-Hep-1, JHH-4, HLF, RBE, SSP-25) were generated by fluorescence 2-dimensional difference gel electrophoresis. In fluorescence 2-dimensional difference gel electrophoresis, proteins are labeled with fluorescent dyes before electrophoresis for more accurate quantitative expression analysis. We identified 11 protein spots that distinguished AFP-producing cell lines from nonproducing cell lines by multivariate studies. The spots showed consistent alterations in amount in AFP-producing cell lines (6 up-regulated and 5 down-regulated). An additional 5 liver cancer cell lines (KIM-1, KYN-2, KYN-3, PH5-CH, PH5-T) also were correctly grouped with respect to their AFP production on the basis of the intensity of the 11 protein spots. The proteins corresponding to the 11 selected spots were identified by mass spectrometry and were categorized into 4 groups based on their known role in apoptosis, glucose metabolism, cytoskeletal organization, or translation. In conclusion, we found a novel association of AFP with other proteins. Their interaction should provide insight into the biology of AFP-producing hepatocellular carcinoma cells.
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Affiliation(s)
- Hideki Yokoo
- Cancer Proteomics Project, National Cancer Center Research Institute, Tokyo, Japan
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Chen HY, Tang NH, Li XJ, Zhang SJ, Chen ZX, Wang XZ. Transfection and expression of hepatitis B virus x gene and its effect on apoptosis in HL-7702 cells. World J Gastroenterol 2004; 10:959-64. [PMID: 15052674 PMCID: PMC4717112 DOI: 10.3748/wjg.v10.i7.959] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To investigate the effects of hepatitis B virus x gene and its protein product HBxAg on apoptosis in hepatocyte line HL-7702.
METHODS: The reconstituted plasmid pcDNA3-x was established through recombination DNA technique; pcDNA3-X was transfected into HL-7702 cells by lipid-mediated trasfection. Positive clones were screened by G418, and HL-7702/HBx cells were analysed by the RT-PCR to confirm the steady expression of X gene in HL-7702 cells. The apoptosis rate in HL-7702 cells was determined by flow cytometry, TUNEL technology, electronic microscope. At the mean time, pcDNA3-X was transfected transiently into HL-7702 cells, and total RNA from HL-7702 cells was extracted 24, 48, 72, 96 and 120 h after the transient transfection, and semi-quantitative analysis was performed by RT-PCR to detect the expression of HBV X gene. Furthermore, apoptosis rate in HL-7702 cells was determined by flow cytometry analysis at the different times.
RESULTS: RT-PCR analysis showed that HBV X gene could be expressed stably in HL-7702 cells. Both flow cytometry and TUNEL technology revealed that the apoptosis rates of HL-7702/HBx cells were much higher than those of HL-7702/ pcDNA3 and HL-7702 cells. Furthermore, the apoptotic phenomena and apoptotic body were observed in HL-7702/ HBx cells under electronic microscope, but not in HL-7702/ pcDNA3 and HL-7702 cells. In the experiment of transient transfection, RT-PCR reveals that X gene was expressed most at 72 h after transfection; and the apoptosis rate reached the highest at the same time. After that, the apoptosis rate was reduced with the decrease of the X gene expression.
CONCLUSION: HBV X gene and X protein can promote the apoptosis in hepatocyte. And there exist a quantity-effect relationship between the X gene expression and apoptosis rate in hepatocyte.
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Affiliation(s)
- Hong-Ying Chen
- Department of Gastroenterology, Union Hospital, Fujian Medical University, Fuzhou 350001, Fujian Province, China.
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Chen HY, Tang NH, Zhang SJ, Chen ZX, Wang XZ. Construction of hepatitis B virus X gene expression vector in eucaryotic cells and its transfection in HL-7702 cells. Shijie Huaren Xiaohua Zazhi 2004; 12:614-617. [DOI: 10.11569/wcjd.v12.i3.614] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To establish a human hepatocyte cell line which can express hepatitis B virus (HBV) X gene.
METHODS: HBV X gene was obtained through PCR techn-ology. After A-tailing added, X gene was connected into vector PUCmT. Vector PUCmT-X and PcDNA3 were digested with EcoRI and HindIII. The fragments of X and PcDNA3 were connected to establish reconstituted plasmid PcDNA3-X. Then PcDNA3-X and PcDNA3 were transfected into HL-7702 cells by lipid-mediated transfection. After selected with G418, HL-7702/HBx cells were analysed by the reverse transcription-PCR to confirm the steady expression of X gene in HL-7702.
RESULTS: Reconstituted plasmid PcDNA3-X included the anticipated fragment of HBV X gene was proved by auto-sequencing assay. RT-PCR analysis showed that reconstituted plasmid PcDNA3-X could express the X protein efficiently in HL-7702 cells.
CONCLUSION: Hepatocyte can express HBV X gene, which is an ideal model to study the effect of HBV X gene on the development of hepatitis and hepatocelular carcinoma.
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Disson O, Haouzi D, Desagher S, Loesch K, Hahne M, Kremer EJ, Jacquet C, Lemon SM, Hibner U, Lerat H. Impaired clearance of virus-infected hepatocytes in transgenic mice expressing the hepatitis C virus polyprotein. Gastroenterology 2004; 126:859-72. [PMID: 14988840 DOI: 10.1053/j.gastro.2003.12.005] [Citation(s) in RCA: 57] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
BACKGROUND & AIMS Multiple molecular mechanisms are likely to contribute to the establishment of persistent infection by hepatitis C virus (HCV). The aim of this work was to study the evasion of cell-mediated antiviral immune responses in transgenic mice with liver-targeted expression of the hepatitis C viral genome. These mice develop steatosis and hepatocellular carcinoma and constitute a murine model of chronic HCV infection. METHODS Mice of the FL-N/35 lineage were infected with replication-deficient adenoviral vectors encoding beta-galactosidase, and the persistence of infected cells was measured by histochemistry and enzymatic assays. RESULTS Hepatocytes from the HCV(+) transgenic mice are deficient in eliminating an adenoviral infection, despite an apparently normal T-cell response. The defect in adenoviral clearance was associated with resistance of transgenic hepatocytes to apoptosis induced by Fas/APO1/CD95 death receptor stimulation, a major pathway of cell killing by cytotoxic T lymphocytes. The attenuation of Fas-mediated apoptosis observed in the murine model was associated with a reduced abundance of Bid, a BH3-only member of the Bcl-2 family of apoptosis regulators. CONCLUSIONS Our results suggest that viral evasion of cell-mediated immune responses leading to apoptotic death of hepatocytes may contribute to viral persistence. Such a mechanism might also contribute to the development of liver cancer in HCV.
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Affiliation(s)
- Olivier Disson
- Institut de Génétique Moléculaire, Centre National Recherche Scientifique, Unite Mixte de Recherche 5535, IFR 122, Montpelier, France
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Bernstein H, Payne CM, Kunke K, Crowley-Weber CL, Waltmire CN, Dvorakova K, Holubec H, Bernstein C, Vaillancourt RR, Raynes DA, Guerriero V, Garewal H. A proteomic study of resistance to deoxycholate-induced apoptosis. Carcinogenesis 2004; 25:681-92. [PMID: 14729586 DOI: 10.1093/carcin/bgh072] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
The development of apoptosis resistance appears to be an important factor in colon carcinogenesis. To gain an understanding of the molecular pathways altered during the development of apoptosis resistance, we selected three cell lines for resistance to induction of apoptosis by deoxycholate, an important etiologic agent in colon cancer. We then evaluated gene expression levels for 825 proteins in these resistant lines, compared with a parallel control line not subject to selection. Eighty-two proteins were identified as either over-expressed or under-expressed in at least two of the resistant lines, compared with the control. Thirty-five of the 82 proteins (43%) proved to have a known role in apoptosis. Of these 35 proteins, 21 were over-expressed and 14 were under-expressed. Of those that were over-expressed 18 of 21 (86%) are anti-apoptotic in some circumstances, of those that were under-expressed 11 of 14 (79%) are pro-apoptotic in some circumstances. This finding suggests that apoptosis resistance during selection among cultured cells, and possibly in the colon during progression to cancer, may arise by constitutive over-expression of multiple anti-apoptotic proteins and under-expression of multiple pro-apoptotic proteins. The major functional groups in which altered expression levels were found are post-translational modification (19 proteins), cell structure (cytoskeleton, microtubule, actin, etc.) (17 proteins), regulatory processes (11 proteins) and DNA repair and cell cycle checkpoint mechanisms (10 proteins). Our findings, overall, bear on mechanisms by which apoptosis resistance arises during progression to colon cancer and suggest potential targets for cancer treatment. In addition, assays of normal-appearing mucosa of colon cancer patients, for over- or under-expression of genes found to be altered in our resistant cell lines, may allow identification of early biomarkers of colon cancer risk.
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Affiliation(s)
- Harris Bernstein
- Department of Microbiology and Immunology, Arizona Cancer Center, Tucson, AZ 85724, USA
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44
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Chen GG, Merchant JL, Lai PBS, Ho RLK, Hu X, Okada M, Huang SF, Chui AKK, Law DJ, Li YG, Lau WY, Li AKC. Mutation of p53 in recurrent hepatocellular carcinoma and its association with the expression of ZBP-89. THE AMERICAN JOURNAL OF PATHOLOGY 2003; 162:1823-9. [PMID: 12759240 PMCID: PMC1868140 DOI: 10.1016/s0002-9440(10)64317-9] [Citation(s) in RCA: 41] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
p53 has recently been identified as a downstream target of ZBP-89, a zinc finger transcription factor. ZBP-89 promotes growth arrest through stabilization of the p53 protein. The aim of this study is to determine the status of the p53 gene in recurrent human hepatocellular carcinoma (HCC) and test the link between the expression of ZBP-89 and the p53 gene. The results showed that mutations in the p53 gene were frequently detected in recurrent HCC. The interval between surgical resection and the recurrence of HCC was significantly longer in patients with the wild-type p53 gene than those with mutations, strongly suggesting a pathological role for the mutant p53 gene in HCC recurrence. Among those positive for the p53 protein, nearly 85% (18 of 21) showed nuclear localization of the p53 protein while only about 14% (3 of 21) were positive for the p53 protein in the cytoplasm. ZBP-89 co-localized with p53 in the nucleus in about 67% (12 of 18) of all cases positive for the nuclear p53 protein, suggesting that ZBP-89 may play a role in the nuclear accumulation of the p53 protein in a subset of recurrent HCC. With accumulation of p53 protein in the nucleus, tumor cells undergo apoptosis and thus are more susceptible to radiotherapy and chemotherapy. Therefore, co-localization of p53 protein with ZBP-89 may define a subgroup of recurrent HCC that is more sensitive to treatment.
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Affiliation(s)
- George G Chen
- Department of Surgery and the Sir Y. K. Pao Center for Cancer, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong.
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Hsu EC, Hsi B, Hirota-Tsuchihara M, Ruland J, Iorio C, Sarangi F, Diao J, Migliaccio G, Tyrrell DL, Kneteman N, Richardson CD. Modified apoptotic molecule (BID) reduces hepatitis C virus infection in mice with chimeric human livers. Nat Biotechnol 2003; 21:519-25. [PMID: 12704395 DOI: 10.1038/nbt817] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2002] [Accepted: 01/30/2003] [Indexed: 11/09/2022]
Abstract
Hepatitis C virus (HCV) encodes a polyprotein consisting of core, envelope (E1, E2, p7), and nonstructural polypeptides (NS2, NS3, NS4A, NS4B, NS5A, NS5B). The serine protease (NS3/NS4A), helicase (NS3), and polymerase (NS5B) constitute valid targets for antiviral therapy. We engineered BH3 interacting domain death agonist (BID), an apoptosis-inducing molecule, to contain a specific cleavage site recognized by the NS3/NS4A protease. Cleavage of the BID precursor molecule by the viral protease activated downstream apoptotic molecules of the mitochondrial pathway and triggered cell death. We extended this concept to cells transfected with an infectious HCV genome, hepatocytes containing HCV replicons, a Sindbis virus model for HCV, and finally HCV-infected mice with chimeric human livers. Infected mice injected with an adenovirus vector expressing modified BID exhibited HCV-dependent apoptosis in the human liver xenograft and considerable declines in serum HCV titers.
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Affiliation(s)
- Eric C Hsu
- Ontario Cancer Institute (Advanced Medical Discoveries Institute), 620 University Ave., Suite 706, Toronto, ON M5G 2C1, Canada
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