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Han SR, Lee CH, Im JY, Kim JH, Kim JH, Kim SJ, Cho YW, Kim E, Kim Y, Ryu JH, Ju MH, Jeong JS, Lee SW. Targeted suicide gene therapy for liver cancer based on ribozyme-mediated RNA replacement through post-transcriptional regulation. MOLECULAR THERAPY. NUCLEIC ACIDS 2020; 23:154-168. [PMID: 33335800 PMCID: PMC7732968 DOI: 10.1016/j.omtn.2020.10.036] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/13/2020] [Accepted: 10/24/2020] [Indexed: 12/16/2022]
Abstract
Hepatocellular carcinoma (HCC) has high fatality rate and limited therapeutic options. Here, we propose a new anti-HCC approach with high cancer-selectivity and efficient anticancer effects, based on adenovirus-mediated Tetrahymena group I trans-splicing ribozymes specifically inducing targeted suicide gene activity through HCC-specific replacement of telomerase reverse transcriptase (TERT) RNA. To confer potent anti-HCC effects and minimize hepatotoxicity, we constructed post-transcriptionally enhanced ribozyme constructs coupled with splicing donor and acceptor site and woodchuck hepatitis virus post-transcriptional regulatory element under the control of microRNA-122a (miR-122a). Adenovirus encoding post-transcriptionally enhanced ribozyme improved trans-splicing reaction and decreased human TERT (hTERT) RNA level, efficiently and selectively retarding hTERT-positive liver cancers. Adenovirus encoding miR-122a-regulated ribozyme caused selective liver cancer cytotoxicity, the efficiency of which depended on ribozyme expression level relative to miR-122a level. Systemic administration of adenovirus encoding the post-transcriptionally enhanced and miR-regulated ribozyme caused efficient anti-cancer effects at a single dose of low titers and least hepatotoxicity in intrahepatic multifocal HCC mouse xenografts. Minimal liver toxicity, tissue distribution, and clearance pattern of the recombinant adenovirus were observed in normal animals administered either systemically or via the hepatic artery. Post-transcriptionally regulated RNA replacement strategy mediated by a cancer-specific ribozyme provides a clinically relevant, safe, and efficient strategy for HCC treatment.
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Affiliation(s)
- Seung Ryul Han
- R&D Center, Rznomics, Inc., Seongnam 13486, Republic of Korea
| | - Chang Ho Lee
- Department of Life Convergence, Research Institute of Advanced Omics, Dankook University, Yongin 16890, Republic of Korea
| | - Ji Young Im
- Department of Life Convergence, Research Institute of Advanced Omics, Dankook University, Yongin 16890, Republic of Korea
| | - Ju Hyun Kim
- Department of Life Convergence, Research Institute of Advanced Omics, Dankook University, Yongin 16890, Republic of Korea
| | - Ji Hyun Kim
- R&D Center, Rznomics, Inc., Seongnam 13486, Republic of Korea
| | - Sung Jin Kim
- Department of Life Convergence, Research Institute of Advanced Omics, Dankook University, Yongin 16890, Republic of Korea
| | - Young Woo Cho
- New Drug Development Center, Osong Medical Innovation Foundation, Cheongju 28160, Republic of Korea.,College of Pharmacy, Chungbuk National University, Cheongju 28644, Republic of Korea
| | - Eunkyung Kim
- New Drug Development Center, Osong Medical Innovation Foundation, Cheongju 28160, Republic of Korea.,College of Pharmacy, Chungbuk National University, Cheongju 28644, Republic of Korea
| | - Youngah Kim
- New Drug Development Center, Osong Medical Innovation Foundation, Cheongju 28160, Republic of Korea
| | - Ji-Ho Ryu
- New Drug Development Center, Osong Medical Innovation Foundation, Cheongju 28160, Republic of Korea.,College of Pharmacy, Chungbuk National University, Cheongju 28644, Republic of Korea
| | - Mi Ha Ju
- Department of Pathology and Immune-network Pioneer Research Center, Dong-A University College of Medicine, Busan 602-714, Republic of Korea
| | - Jin Sook Jeong
- Department of Pathology and Immune-network Pioneer Research Center, Dong-A University College of Medicine, Busan 602-714, Republic of Korea
| | - Seong-Wook Lee
- R&D Center, Rznomics, Inc., Seongnam 13486, Republic of Korea.,Department of Life Convergence, Research Institute of Advanced Omics, Dankook University, Yongin 16890, Republic of Korea
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Ezzat WM, Amr KS, Raouf HA, Elhosary YA, Hegazy AE, Fahim HH, Kamel RR. Relationship Between Serum microRNA155 and Telomerase Expression in Hepatocellular Carcinoma. Arch Med Res 2016; 47:349-355. [PMID: 27751368 DOI: 10.1016/j.arcmed.2016.08.003] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2016] [Accepted: 08/04/2016] [Indexed: 02/07/2023]
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Kim YH, Kim KT, Lee SJ, Hong SH, Moon JY, Yoon EK, Kim S, Kim EO, Kang SH, Kim SK, Choi SI, Goh SH, Kim D, Lee SW, Ju MH, Jeong JS, Kim IH. Image-aided Suicide Gene Therapy Utilizing Multifunctional hTERT-targeting Adenovirus for Clinical Translation in Hepatocellular Carcinoma. Am J Cancer Res 2016; 6:357-68. [PMID: 26909111 PMCID: PMC4737723 DOI: 10.7150/thno.13621] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2015] [Accepted: 11/21/2015] [Indexed: 02/06/2023] Open
Abstract
Trans-splicing ribozyme enables to sense and reprogram target RNA into therapeutic transgene and thereby becomes a good sensing device for detection of cancer cells, judging from transgene expression. Previously we proposed PEPCK-Rz-HSVtk (PRT), hTERT targeting trans-splicing ribozyme (Rz) driven by liver-specific promoter phosphoenolpyruvate carboxykinase (PEPCK) with downstream suicide gene, herpes simplex virus thymidine kinase (HSVtk) for hepatocellular carcinoma (HCC) gene therapy. Here, we describe success of a re-engineered adenoviral vector harboring PRT in obtaining greater antitumor activity with less off-target effect for clinical application as a theranostics. We introduced liver-selective apolipoprotein E (ApoE) enhancer to the distal region of PRT unit to augment activity and liver selectivity of PEPCK promoter, and achieved better transduction into liver cancer cells by replacement of serotype 35 fiber knob on additional E4orf1-4 deletion of E1&E3-deleted serotype 5 back bone. We demonstrated that our refined adenovirus harboring PEPCK/ApoE-Rz-HSVtk (Ad-PRT-E) achieved great anti-tumor efficacy and improved ability to specifically target HCC without damaging normal hepatocytes. We also showed noninvasive imaging modalities were successfully employed to monitor both how well a therapeutic gene (HSVtk) was expressed inside tumor and how effectively a gene therapy took an action in terms of tumor growth. Collectively, this study suggests that the advanced therapeutic adenoviruses Ad-PRT-E and its image-aided evaluation system may lead to the powerful strategy for successful clinical translation and the development of clinical protocols for HCC therapy.
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Jebar AH, Vile RG, Melcher AA, Griffin S, Selby PJ, Errington-Mais F. Progress in clinical oncolytic virus-based therapy for hepatocellular carcinoma. J Gen Virol 2015; 96:1533-50. [DOI: 10.1099/vir.0.000098] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
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LI HONGSHENG, WANG QIAOYU, LI DONGLI, WANG QUANSHI, WU HUBING. Combination of HSV1-TK/shTERT by retrovirus vector inhibits hepatocellular carcinoma cell growth in vitro and in vivo. Oncol Rep 2014; 33:1307-13. [DOI: 10.3892/or.2014.3697] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2014] [Accepted: 11/20/2014] [Indexed: 11/06/2022] Open
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Shiraha H, Yamamoto K, Namba M. Human hepatocyte carcinogenesis (review). Int J Oncol 2013; 42:1133-8. [PMID: 23426905 PMCID: PMC3622653 DOI: 10.3892/ijo.2013.1829] [Citation(s) in RCA: 98] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2012] [Accepted: 10/22/2012] [Indexed: 02/07/2023] Open
Abstract
Hepatocellular carcinoma is the third most frequent cause of cancer-related death worldwide; and its incidence rate is increasing. Clinical and molecular medical analyses have revealed substantial information on hepatocarcinogenesis. Hepatocarcinogenesis is a stepwise process during which multiple genes are altered. Genetic changes and their biological consequences in human HCC can be divided into at least 4 groups: i) tumor suppressor genes (p53, retinoblastoma, phosphatase tensin homolog and runt-related transcription factor 3), ii) oncogenes (myc, K-ras, BRAF), iii) reactivation of developmental pathways (Wnt, hedgehog), and iv) growth factors and their receptors (transforming growth factor-α, insulin-like growth factor-2 receptor). An experimental model of human hepatocarcinogenesis such as in vitro neoplastic transformation of human hepatocytes has not been successfully achieved yet, but several immortalized human hepatocyte cell lines have been established. These immortalized human hepatocytes will become useful tools for the elucidation of hepatocarcinogenesis, especially for the initial step of multistep hepatocarcinogenesis.
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Affiliation(s)
- Hidenori Shiraha
- Department of Gastroenterology and Hepatology, Okayama University Faculty of Medicine, Okayama 700-8558, Japan.
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Lyra-González I, Flores-Fong LE, González-García I, Medina-Preciado D, Armendáriz-Borunda J. Adenoviral gene therapy in hepatocellular carcinoma: a review. Hepatol Int 2012. [DOI: 10.1007/s12072-012-9367-2] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
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Hu Y, Shen Y, Ji B, Wang L, Zhang Z, Zhang Y. Combinational RNAi gene therapy of hepatocellular carcinoma by targeting human EGFR and TERT. Eur J Pharm Sci 2011; 42:387-91. [PMID: 21238587 DOI: 10.1016/j.ejps.2011.01.004] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2010] [Revised: 12/29/2010] [Accepted: 01/06/2011] [Indexed: 10/18/2022]
Abstract
Both human telomerase reverse transcriptase (hTERT) and epidermal growth factor receptor (hEGFR) are ideal targets for RNA interference (RNAi)-based gene therapy of hepatocellular carcinoma. Two shRNA expression plasmids pU6-shTERT and pU6-shEGFR targeting hTERT and hEGFR, respectively, were separately formulated as pegylated immuno-lipopolyplexes, a novel non-viral gene delivery system. In vitro studies showed that when pU6-shTERT and pU6-shEGFR were combined and applied to SMMC-7721 cells, there was a significant additive effect on cytotoxicity as well as cell apoptosis, compared to pU6-shTERT or pU6-shEGFR alone, with a cell viability of 50.9±7.4%, 79.2±3.6% and 77.1±3.6%, respectively, and with a cell apoptotic rate of 44.8±0.9%, 25.1±0.4% and 29.5±0.8%, respectively. In vivo study in SMMC-7721 xenograft tumor model demonstrated that intravenous administration of PILP-formulated pU6-shTERT and pU6-shEGFR caused an additive effect on tumor growth inhibition, compared to pU6-shTERT or pU6-shEGFR alone, with a tumor growth inhibition rate of 74.0%, 36.3% and 46.1%, respectively, which is consistent with the downregulated EGFR and TERT mRNA expression. The results suggest that combinational RNAi gene therapy of hepatocellular carcinoma by targeting human EGFR and TERT with pegylated immuno-lipopolyplexes is a new and good strategy.
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Affiliation(s)
- Yurong Hu
- School of Pharmacy, Zhengzhou University, 100 Science Road, Zhengzhou 450001, Henan, PR China
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Liver-specific gene therapy of hepatocellular carcinoma by targeting human telomerase reverse transcriptase with pegylated immuno-lipopolyplexes. Eur J Pharm Biopharm 2011; 78:320-5. [PMID: 21220007 DOI: 10.1016/j.ejpb.2010.12.036] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2010] [Revised: 12/11/2010] [Accepted: 12/16/2010] [Indexed: 11/21/2022]
Abstract
The purpose of this study is to explore the possibility and feasibility of liver-specific gene therapy. A shRNA expression plasmid against human telomerase reverse transcriptase (hTERT) was constructed under the control of liver-specific promoter apolipoprotein A-I (ApoAI), designated as pApoAI-shTERT, and its liver-specific cytotoxicity and inhibition of telomerase activity were first evaluated in different cell lines, and its therapeutic effect was further studied in SMMC-7721 human liver tumor-bearing mice in vivo. The results showed that compared to pU6-shTERT, a shRNA expression plasmid against hTERT under the control of U6 promoter, pApoAI-shTERT only significantly diminished the cell viability in the telomerase positive hepatocarcinoma cells and showed no cytotoxicity in the telomerase negative cell lines as well as in the telomerase positive cell line of non-liver origin. Besides, pApoAI-shTERT only significantly reduced telomerase activity in the telomerase positive cell lines of liver origin. Intravenous administration of pegylated immuno-lipopolyplexes (PILP) formulated green fluorescent protein (GFP) expression plasmid under the control of ApoAI into liver tumor-bearing mice resulted in restricted GFP expression in liver and liver tumor. The treatment of pApoAI-shTERT formulated as PILP caused a 56% increase in the life span of SMMC-7721 tumor-bearing mice in vivo relative to the control, which was in agreement with the reduced tumor size and down-regulated hTERT mRNA level in the tumors. We conclude that the vector pApoAI-shTERT was able to cause liver-specific and hTERT target-specific cytotoxicity, and utilizing PILP to deliver pApoAI-shTERT is a promising strategy for liver-specific gene therapy.
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Ageing, telomeres, senescence, and liver injury. J Hepatol 2010; 53:950-61. [PMID: 20739078 DOI: 10.1016/j.jhep.2010.06.009] [Citation(s) in RCA: 132] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/03/2010] [Revised: 06/24/2010] [Accepted: 06/26/2010] [Indexed: 02/08/2023]
Abstract
Populations in developed countries continue to grow older and an understanding of the ageing process to allow healthy ageing carries important medical implications. Older individuals are more susceptible to most acquired liver disorders and more vulnerable to the consequences of liver disease. Accordingly, age is a critical determinant of outcome for hepatitis C virus infection and liver transplantation. In this review we describe changes in the ageing liver and discuss mechanisms of senescence at the cellular level. In particular, we focus on mechanisms by which inflammation, oxidative stress, and oncogenic stress accelerate cellular senescence. In the setting of chronic hepatic injury and inflammation, cellular senescence functions as an essential stress-response mechanism to limit the proliferation of damaged cells and reduce the risk of malignancy, but this benefit is achieved at the expense of senescence-related organ dysfunction. The dual role of cell senescence in chronic liver disease will make this an intriguing but challenging area for future clinical interventions.
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Sturgeon CM, Duffy MJ, Hofmann BR, Lamerz R, Fritsche HA, Gaarenstroom K, Bonfrer J, Ecke TH, Grossman HB, Hayes P, Hoffmann RT, Lerner SP, Löhe F, Louhimo J, Sawczuk I, Taketa K, Diamandis EP. National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines for use of tumor markers in liver, bladder, cervical, and gastric cancers. Clin Chem 2010; 56:e1-48. [PMID: 20207771 DOI: 10.1373/clinchem.2009.133124] [Citation(s) in RCA: 139] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
BACKGROUND Updated National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines for the use of tumor markers in the clinic have been developed. METHODS Published reports relevant to use of tumor markers for 4 cancer sites--liver, bladder, cervical, and gastric--were critically reviewed. RESULTS Alpha-fetoprotein (AFP) may be used in conjunction with abdominal ultrasound for early detection of hepatocellular carcinoma (HCC) in patients with chronic hepatitis or cirrhosis associated with hepatitis B or C virus infection. AFP concentrations >200 microg/L in cirrhotic patients with typical hypervascular lesions >2 cm in size are consistent with HCC. After a diagnosis of HCC, posttreatment monitoring with AFP is recommended as an adjunct to imaging, especially in the absence of measurable disease. Although several urine markers have been proposed for bladder cancer, none at present can replace routine cystoscopy and cytology in the management of patients with this malignancy. Some may, however, be used as complementary adjuncts to direct more effective use of clinical procedures. Although carcinoembryonic antigen and CA 19-9 have been proposed for use gastric cancer and squamous cell carcinoma antigen for use in cervical cancer, none of these markers can currently be recommended for routine clinical use. CONCLUSIONS Implementation of these recommendations should encourage optimal use of tumor markers for patients with liver, bladder, cervical, or gastric cancers.
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Affiliation(s)
- Catharine M Sturgeon
- Department of Clinical Biochemistry, Royal Infirmary of Edinburgh, Edinburgh, UK.
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Effendi K, Mori T, Komuta M, Masugi Y, Du W, Sakamoto M. Bmi-1 gene is upregulated in early-stage hepatocellular carcinoma and correlates with ATP-binding cassette transporter B1 expression. Cancer Sci 2010; 101:666-72. [PMID: 20085590 PMCID: PMC11158551 DOI: 10.1111/j.1349-7006.2009.01431.x] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Overexpression of "stemness gene"Bmi-1 has been identified in some solid tumors. We investigated Bmi-1 expression in hepatocellular carcinoma (HCC) and ATP-binding cassette transporter B1 (ABCB1) as a new potential target for Bmi-1. Bmi-1 was highly expressed in HCC cell lines and the most well differentiated cell line, KIM-1, showed the highest expression. Immunohistochemical, immunocytochemical, and immunoelectron microscopic analysis showed the Bmi-1 protein as having a high intensity of small dots within the nucleus which reflected concentrated sites of Bmi-1 repressive activity. Clear "dot-pattern" staining was observed in 24 of 37 (65%) well differentiated HCC (including 13 of 21 early nodules [62%]), in 32 of 71 (45%) moderately differentiated HCC, and 7 of 14 (50%) poorly differentiated HCC. A similar expression was not observed in non-cancerous background regions. High Bmi-1 expression was observed in the early and well differentiated HCC. Furthermore, overexpression and suppression of Bmi-1 was followed by a respective increase and decrease in ABCB1 expression. As with Bmi-1, high ABCB1 expression was also observed in the early and well differentiated HCC. A strong correlation between ABCB1 and Bmi-1 mRNA expression was seen in HCC cell lines and clinical samples (Pearson's correlation coefficient 0.95 and 0.90, respectively). The Bmi-1 gene is upregulated in HCC, and in particular is highly expressed in early and well differentiated HCC. The fact that this expression correlated with that of ABCB1 suggests a new regulation target for Bmi-1, and gives new insight into early hepatocarcinogenesis mechanisms and potential targets for future HCC treatment.
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Affiliation(s)
- Kathryn Effendi
- Department of Pathology, School of Medicine, Keio University, Tokyo, Japan
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Nishikawa T, Nakajima T, Katagishi T, Okada Y, Jo M, Kagawa K, Okanoue T, Itoh Y, Yoshikawa T. Oxidative stress may enhance the malignant potential of human hepatocellular carcinoma by telomerase activation. Liver Int 2009; 29:846-56. [PMID: 19141026 DOI: 10.1111/j.1478-3231.2008.01963.x] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
Abstract
BACKGROUND/AIMS Continuous oxidative stress (OS) plays an important role in the progression of chronic liver diseases and hepatocarcinogenesis through telomere shortening in hepatocytes. However, it has not been established how the OS influences the progression of human hepatocellular carcinomas (HCCs). We examined the correlations of OS with telomere length of cancer cells, telomerase activity and other clinicopathological factors in 68 HCCs. METHODS The level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a marker of OS was examined immunohistochemically and OS was scored in four grades (0-3). The telomere length of cancer cells was measured by quantitative fluorescence in situ hybridization. Telomerase activity was measured by (i) immunodetection of human telomerase reverse transcriptase (hTERT) and (ii) telomere repeat amplification protocol (TRAP) assay. Telomerase related proteins, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and Akt, and other clinicopathological factors were also evaluated. RESULTS As the OS grade increased, the average telomere length became significantly shorter in HCCs, especially in the hTERT-negative group. In the state of high-grade OS, hTERT-positive HCC cells showed more proliferative and less apoptotic features compared with hTERT-negative HCC cells. Telomerase activity, as measured by the TRAP assay, was strongly correlated with OS grade in HCCs. Furthermore, a high OS grade was correlated with the downexpression of PTEN and the activation of Akt. CONCLUSIONS Oxidative stress enhanced the malignant potential of HCCs through the activation of telomerase, which raises the possibility of using OS as a marker for assessing the clinical state of HCCs.
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Affiliation(s)
- Taichiro Nishikawa
- Kyoto Prefectural University of Medicine Graduate School of Medical Science, Molecular Gastroenterology and Hepatology, Kyoto, Japan.
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Wong CH, Chan SKP, Chan HLY, Tsui SKW, Feitelson M. The Molecular Diagnosis of Hepatitis B Virus-Associated Hepatocellular Carcinoma. Crit Rev Clin Lab Sci 2008; 43:69-101. [PMID: 16531275 DOI: 10.1080/10408360500410407] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC) worldwide. The pathogenesis of HBV-associated HCC has been studied extensively, and molecular changes during malignant transformation have been identified. It has been proposed that the insertion of HBV DNA into the human genome results in chromosomal instability and inactivation of tumor suppressor genes. Transactivation of oncogenes, inactivation of tumor suppressor genes, and alteration of the cell cycle by HBV proteins are also involved in the progression of hepatocellular carcinogenesis. Traditional clinical examinations of HCC, such as biopsy, computer tomography, ultrasonic imaging, and detection of such biomarkers as a-fetoprotein, are currently the "gold standard" in diagnosis. These tests diagnose HCC only in the late stages of disease. This limitation has greatly reduced the chance of survival of HCC patients. To resolve this problem, new biomarkers that can diagnose HCC in earlier stages are necessary. Based on recent molecular studies of the effects of HBV on cellular transformation, differentially expressed biomarkers of HBV infection have been elucidated. With the analyses of the HBV replication profile, the viral load (HBV DNA levels) of patients, and the viral protein expression, the severity of hepatitis in the preneoplastic stages can be assessed. In the future, with the molecular profiles identified by genomic and proteomic approaches, stage-specific biomarkers should be identified to monitor the progression and prognosis of HCC.
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Affiliation(s)
- Chi-Hang Wong
- Center for Emerging Infectious Diseases, The Chinese University, Hong Kong, Shatin, N.T., Hong Kong SAR, China
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High telomerase activity and long telomeres in advanced hepatocellular carcinomas with poor prognosis. J Transl Med 2008; 88:144-52. [PMID: 18158557 DOI: 10.1038/labinvest.3700710] [Citation(s) in RCA: 77] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Telomerase reactivation and telomere maintenance are crucial in carcinogenesis and tumor progression. In this study, the relationships between telomere parameters, chromosomal instability and clinicopathological features were evaluated in hepatocellular carcinomas (HCCs). Telomere length (TL), telomerase activity (TA) and human telomerase reverse transcriptase (hTERT) mRNA levels were measured in 49 hepatitis B virus (HBV)-related HCCs and corresponding non-tumorous tissues. The results were compared with clinicopathological data, including differentiation, multipolar mitosis (MM), anaphase bridge, immunohistochemical stain results for cytokeratin 19 (CK19) and patient outcome. TL of HCCs ranged from 4.7 to 13.1 kb, and 44.4% of HCCs showed telomere lengthening. hTERT mRNA levels and TA were closely related (P=0.008), and were significantly higher in HCCs than non-tumorous tissues. TL was significantly higher in HCCs with strong TA (P=0.048), high hTERT mRNA levels (P=0.001) and poor differentiation (P=0.041). Frequent MM was associated with poor differentiation (P=0.007) and advanced stage (P<0.001). TA was positively correlated with MM, anaphase bridges and advanced stage (P=0.019, P=0.017 and P=0.029). Thirteen (28.3%) HCCs were CK19+ and demonstrated longer telomeres than CK19- HCCs (P=0.046). Overall survival was poor in HCCs with MM >0.4 per field (P=0.016), high TA (P=0.009) and high TL ratio (HCC/non-HCC) >0.8 (P=0.044). Our results show that long telomeres, high TA and high mitotic instability are poor prognostic markers for HBV-related HCCs and their close association suggests that telomere maintenance may be important for the progression of HCCs with high chromosomal instability to more aggressive ones.
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Bu X, Jia F, Wang W, Guo X, Wu M, Wei L. Coupled down-regulation of mTOR and telomerase activity during fluorouracil-induced apoptosis of hepatocarcinoma cells. BMC Cancer 2007; 7:208. [PMID: 17996122 PMCID: PMC2186345 DOI: 10.1186/1471-2407-7-208] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2007] [Accepted: 11/12/2007] [Indexed: 11/16/2022] Open
Abstract
Background Hepatocellular carcinoma (HCC) is the most invasive and frequently diagnosed malignancy and the second leading cause of cancer death in many regions of Asia. The PI3K/Akt/mTOR signal pathway is involved in multiple cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. Up-regulation of telomerase activity is thought to be a critical step leading to cell transformation. Methods This study investigated changes in mTOR pathway and telomerase activity in hepatocarcinoma cell line SMMC-7721 treated with chemotherapeutic agent 5-fluorouracil (5-Fu). We detected apoptosis of hepatocarcinoma cells by TUNEL assay. Telomerase activity, hTERT transcription level and p- p70 S6k was demonstrated by the telomeric repeat amplification protocol and silver staining assay, Dual-Luciferase Reporter Assay and Western blot analysis respectively. Results Treating SMMC-7721 cells with 5-Fu leads to apoptosis of the cells, and reduction in telomerase activity, as well as a dramatic reduction in the activated form of p70 S6 kinase, a mTOR substrate. The 5-Fu treatment nearly abolishes transcription of hTERT (the major component of telomerase) mRNA. Treating SMMC-7721 cells with Rapamycin, a specific mTOR inhibitor, significantly reduce hTERT protein level but did not affect hTERT transcription. 5-Fu and rapamycin were synergistic in regards to down-regulation of telomerase activity in hepatocarcinoma cells. Conclusion These results suggest that chemotherapeutic agent 5-Fu may down-regulate telomerase activity at both transcriptional level and PI3K/Akt/mTOR pathway-dependent post-transcriptional level to facilitate hepatocellular carcinoma cell apoptosis.
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Affiliation(s)
- Xinxin Bu
- Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Hospital, Second Military Medical Universisty, 225 Changhai Road, Shanghai 200438, China.
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Satra M, Gatselis N, Iliopoulos D, Zacharoulis D, Dalekos GN, Tsezou A. Real-time quantification of human telomerase reverse transcriptase mRNA in liver tissues from patients with hepatocellular cancer and chronic viral hepatitis. J Viral Hepat 2007; 14:41-7. [PMID: 17212643 DOI: 10.1111/j.1365-2893.2006.00769.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
We determined, for the first time, the human telomerase reverse transcriptase (hTERT) mRNA expression, using real-time quantitative PCR, in liver tissues from patients with hepatocellular cancer (HCC; n = 13), chronic hepatitis B (n = 19) and C (n = 13). Liver tissues from the 45 patients and 17 patients without liver disease in whom liver biopsy was performed during cholecystectomy (control group), were investigated for telomerase activity (TA) and hTERT mRNA expression using the LightCycler technology. TA was detected in all HCC tissues compared with 15.6% of chronic hepatitis (P < 0.001) and none of controls (P < 0.001). TA levels and hTERT mRNA were higher in HCC compared with chronic hepatitis (P < 0.001) and normal livers (P < 0.001). hTERT mRNA expression was correlated with TA (P < 0.05). Chronic hepatitis patients who tested negative for TA and hTERT mRNA had significantly lower disease duration (58 +/- 85 months) compared with those tested positive (144 +/- 50 months; P < 0.05). Detection of TA and quantification of hTERT mRNA expression in liver tissues could be useful and additional markers for HCC diagnosis and may serve as prognostic markers for HCC development in chronic viral hepatitis patients. However, we were not able to draw general conclusions at this moment, as the number of chronic hepatitis patients positive for hTERT mRNA was relatively small. Real-time quantification of hTERT mRNA expression as a diagnostic/prognostic marker in patients with chronic hepatitis B and C and its relationship with hepatocarcinogenesis needs further evaluation.
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MESH Headings
- Adult
- Aged
- Aged, 80 and over
- Biopsy
- Carcinoma, Hepatocellular/enzymology
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/pathology
- Female
- Hepacivirus
- Hepatitis B virus
- Hepatitis B, Chronic/enzymology
- Hepatitis B, Chronic/genetics
- Hepatitis B, Chronic/pathology
- Hepatitis C, Chronic/enzymology
- Hepatitis C, Chronic/genetics
- Hepatitis C, Chronic/pathology
- Humans
- Liver Neoplasms/enzymology
- Liver Neoplasms/genetics
- Liver Neoplasms/pathology
- Male
- Middle Aged
- RNA, Messenger/biosynthesis
- RNA, Messenger/genetics
- Reverse Transcriptase Polymerase Chain Reaction
- Statistics, Nonparametric
- Telomerase/biosynthesis
- Telomerase/genetics
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Affiliation(s)
- M Satra
- Department of Biology, Medical School, University of Thessaly, Larissa, Greece
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19
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Abstract
Hepatocellular carcinoma is among the most lethal and prevalent cancers in the human population. Despite its significance, there is only an elemental understanding of the molecular, cellular and environmental mechanisms that drive disease pathogenesis, and there are only limited therapeutic options, many with negligible clinical benefit. This Review summarizes the current state of knowledge of this, the most common and dreaded liver neoplasm, and highlights the principal challenges and scientific opportunities that are relevant to controlling this accelerating global health crisis.
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Affiliation(s)
- Paraskevi A Farazi
- Department of Genetics, Division of Medical Sciences, Harvard University, Boston, Massachusetts 02115, USA
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20
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Abstract
Hepatocellular carcinoma is often diagnosed at an advanced stage, when potentially curative surgical or local ablative therapies are not feasible. There is no effective chemotherapy for hepatocellular carcinoma. Recent advances in cancer biology suggest that a limited number of signalling pathways may be responsible for uncontrolled cell proliferation, the major cellular alteration responsible for the cancer phenotype. Novel anticancer agents target these critical pathways, including the receptor tyrosine kinase pathways, the Wnt/beta-catenin signalling pathway, the ubiquitin/proteasome degradation pathway, the DNA methylation and histone deacetylation pathways, the PI3 kinase/AKT/mTOR pathway, angiogenic pathways, telomerase and the cell cycle. These agents hold promise for improving the outcome of patients with intermediate and advanced hepatocellular carcinoma. Because of the high prevalence of liver cirrhosis in hepatocellular carcinoma patients, to achieve long-term survival of the majority of patients, targeted anticancer therapies will need to be coupled with strategies aimed at reversing the progression of chronic liver disease.
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Affiliation(s)
- Lewis R Roberts
- Mayo Clinic College of Medicine, Miles and Shirley Fiterman Center for Digestive Diseases, 200 First Street SW, Rochester, MN 55905, USA.
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21
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Zhang PH, Zou L, Tu ZG. RNAi-hTERT Inhibition Hepatocellular Carcinoma Cell Proliferation via Decreasing Telomerase Activity. J Surg Res 2006; 131:143-9. [PMID: 16298398 DOI: 10.1016/j.jss.2005.09.017] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2005] [Revised: 08/20/2005] [Accepted: 09/20/2005] [Indexed: 01/06/2023]
Abstract
BACKGROUND RNA interference (RNAi), which has been demonstrated as having great potentional in the fields of gene function and gene therapy, was applied to inhibit the expression of some endogenous genes. Human telomerase reverse transcriptase (hTERT) is highly expressed in hepatocellular carcinoma cells. MATERIALS AND METHODS In combination with DNA vector-based RNAi, quantitative real-time reverse transcription polymerase chain reaction, telomeric repeat amplification protocol-enzyme-linked immunosorbent assay, 3-(4,5 dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay, and xenograft tumor animal techniques, we first constructed three pTZU6 + 1-shRNA-hTERT vectors and their corresponding site-mutated vectors, then transfected them into hepatocarcinoma HepG2, SMMC-7721 cells, and normal liver L02 cells, respectively, injected them into xenograft hepatocarcinoma tumor tissues to induce RNAi, and then detected the alteration of cell and tumor proliferation, telomerase activity, hTERT, and c-myc expression in each treatment. RESULTS The cell proliferation of hepatocellular carcinoma cells both in vitro and in vivo was significantly inhibited by ph1-shRNA, the most effective segment targeted hTERT gene. ph1-shRNA could inhibit telomerase activity, hTERT, and c-myc expression in hepatocarcinoma cells and xenograft tumor tissues compare with the cells treated with empty vector pTZU6 + 1. However, there were no obvious effects on normal liver L02 cells. Moreover, even a single base mutation in siRNAs transcription template would significantly reduce the ability of siRNAs to induce RNA silencing. CONCLUSIONS RNAi-hTERT could inhibit the proliferation of hepatocarcinoma cells specifically via the suppression of telomease activity, hTERT, and c-myc expression. Therefore, hTERT and c-myc play key roles in hepatocarcinoma tumorgenesis, and an RNAi-targeted hTERT strategy would be a potential approach for hepatocarcinoma therapy.
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Affiliation(s)
- Peng-Hui Zhang
- Faculty of Laboratory Medicine in Chongqing University of Medical Sciences, Key Laboratory of Laboratory Medical Diagnosis of Education Ministry, Chongqing China
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22
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Qu ZL, Zou SQ, Cui NQ, Wu XZ, Qin MF, Kong D, Zhou ZL. Upregulation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells. World J Gastroenterol 2005; 11:5627-32. [PMID: 16237755 PMCID: PMC4481478 DOI: 10.3748/wjg.v11.i36.5627] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [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 study the changes of human telomerase reverse transcriptase (hTERT) mRNA expression in human hepatocarcinoma cell lines (HepG2) and cholangiocarcinoma cell lines (QBC939) after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis.
METHODS: HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency. Cells were harvested and total RNA was extracted using TRIzol® reagent. The expression of hTERT mRNA in HepG2 and QBC939 cell lines was assayed by reverse transcription-polymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting.
RESULTS: Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939 cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector. Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939 cells only when transfected with HBx gene.
CONCLUSION: HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactiv-ation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangioca-rcinomas after HBV infection.
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Affiliation(s)
- Zhen-Liang Qu
- Department of Surgery, Tianjin Nankai Hospital, Tianjin 300100, China.
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23
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Varma V, Cohen C. Immunohistochemical and molecular markers in the diagnosis of hepatocellular carcinoma. Adv Anat Pathol 2004; 11:239-49. [PMID: 15322490 DOI: 10.1097/01.pap.0000131822.31576.9d] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Hepatocellular carcinoma (HCC) has distinct morphologic features and can be identified in the majority of cases by routine hematoxylin and eosin (H&E)-stained formalin-fixed paraffin-embedded sections. However, distinguishing a well-differentiated HCC from normal or regenerative tissue may be very difficult in some cases, particularly in small needle aspiration or core biopsies. Furthermore, some of the unusual morphologic variants, including clear-cell, pleomorphic, and sarcomatoid variants, may be mistaken for metastases. Similarly, metastases from various primary tumors to the liver may be mistaken for primary hepatic tumors. In this overview, we summarize the immunohistochemical and molecular markers that have been developed to address these diagnostic challenges. Among the numerous diagnostic markers studied, pCEA, HepPar 1, CD34, CK 7, CK 19, CK 20, and albumin in situ (ISH) have been found to be valuable in distinguishing HCC from metastatic neoplasms of extrahepatic sites.
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Affiliation(s)
- Vijay Varma
- Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.
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24
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Furuta T, Shirai N, Sugimoto M, Ohashi K, Ishizaki T. Pharmacogenomics of proton pump inhibitors. Pharmacogenomics 2004; 5:181-202. [PMID: 15016609 DOI: 10.1517/phgs.5.2.181.27483] [Citation(s) in RCA: 104] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Proton pump inhibitors (PPIs), such as omeprazole, lansoprazole, rabeprazole, esomeprazole, and pantoprazole, are metabolized by cytochrome P450 isoenzyme 2C19 (CYP2C19) in the liver. There are genetic differences that affect the activity of this enzyme. The genotypes of CYP2C19 are classified into three groups: homozygous extensive metabolizer (homEM), heterozygous extensive metabolizer (hetEM), and poor metabolizer (PM). The pharmacokinetics and pharmacodynamics of PPIs differ among the different CYP2C19 genotype groups. Plasma PPI and intragastric pH levels during PPI treatment are the lowest in the homEM group and the highest in the PM group. These CYP2C19 genotype-dependent differences in pharmacokinetics and pharmacodynamics of PPIs are reflected in the cure rates for gastroesophageal reflux disease and Helicobacter pylori infection with PPI-based therapies. The CYP2C19 genotyping test is a useful tool for deciding on the optimal treatment regimen using a PPI, including a dual (PPI plus antibiotic) or a triple (PPI plus two antibiotics) therapy.
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Affiliation(s)
- Takahisa Furuta
- First Department of Medicine, Hamamatsu University School of Medicine, Handa-Yama, Japan.
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25
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Liu SX, Sun WS, Cao YL, Ma CH, Han LH, Zhang LN, Wang ZG, Zhu FL. Antisense oligonucleotide targeting at the initiator of hTERT arrests growth of hepatoma cells. World J Gastroenterol 2004; 10:366-70. [PMID: 14760759 PMCID: PMC4724927 DOI: 10.3748/wjg.v10.i3.366] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [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 evaluate the inhibitory effect of antisense phosphorothioate oligonucleotide (asON) complementary to the initiator of human telomerase catalytic subunit (hTERT) on the growth of hepatoma cells.
METHODS: The as-hTERT was synthesized by using a DNA synthesizer. HepG2.2.15 cells were treated with as-hTERT at the concentration of 10 μmol/L. After 72 h, these cells were obtained for detecting growth inhibition, telomerase activity using the methods of MTT, TRAP-PCR-ELISA, respectively. BALB/c(nu/nu) mice were injected HepG2.2.15 cells and a human-nude mice model was obtained. There were three groups for anti-tumor activity study. Once tumors were established, these animals in the first group were administered as-hTERT and saline. Apoptosis of tumor cells was detected by FCM. In the 2nd group, the animals were injected HepG2.2.15 cells together with as-hTERT. In the third group, the animals were given as-hTERT 24 hours postinjection of HepG2.2.15 cells. The anti-HBV effects were assayed with ELISA in vitro and in vivo.
RESULTS: Growth inhibition was observed in cells treated with as-hTERT in vitro. A significant different in the value of A570 - A630 was found between cells treated with as-hTERT and control (P < 0.01) by MTT method. The telomerase activity of tumor cells treated with as-hTERT was reduced, the value of A450 nm was 0.42 compared to control (1.49) with TRAP-PCR-ELISA. The peak of apoptosis in tumor cells given as-hTERT was 21.12%, but not seen in saline-treated control. A prolonged period of carcinogenesis was observed in the second and third group animals. There was inhibitory effect on the expression of HBsAg and HBeAg in vivo and in vitro.
CONCLUSION: As-hTERT has an anti-tumor activity, which may be useful for gene therapy of tumors.
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Affiliation(s)
- Su-Xia Liu
- Institute of Immunology, Medical School of Shandong University, Wenhua West Road 44, Jinan 250012, Shandong Province, China
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26
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Zhao JM, Li FC, Yu JH, Cui W, Fu BY, Sa WG. Telomerase activity in tuberculous peritonitis and malignant ascites. Shijie Huaren Xiaohua Zazhi 2003; 11:1563-1565. [DOI: 10.11569/wcjd.v11.i10.1563] [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
Abstract
AIM To determine telomerase activity of exfoliated cells in tuberculous peritonitis and malignant ascites, and study the diagnostic value of telomerase activity in differentiating tuberculous peritonitis from malignant ascites.
METHODS TRAP-PCR-ELISA and TRAP-PCR-silver staining were employed to determine telomerase activity in 18 specimens of malignant ascites and 13 specimens of tuberculous peritonitis. Telomerase activities in tuberculous peritonitis and malignant ascites were analysed.
RESULTS Telomerase activity in malignant ascites (0.387±0.023)was significantly higher than that in tuberculous peritonitis(0.023±0.004, P<0.01). The positive rate of telomerase activity in malignant ascites was significantly higher than that in tuberculous peritonitis, 88.9% (16/18) vs 7.7% (1/13), P<0.01. The sensitivity, specificity and accuracy of determination of telomerase activity in diagnosis of malignant ascites were 88.9%, 92.3%, and 90.3%, respectively.
CONCLUSION Telomerase activity is positive in malignant ascites and may serve as a useful indicator for differentiating between tuberculous peritonitis and malignant ascites.
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Affiliation(s)
- Jin-Man Zhao
- Department of Gastroenterology, First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, China
| | - Fu-Cai Li
- Department of Medical Genetics, China Medical University, Shenyang 110001, Liaoning Province, China
| | - Ji-Hong Yu
- Department of Gastroenterology, First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, China
| | - Wei Cui
- Department of Gastroenterology, First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, China
| | - Bao-Yu Fu
- Department of Gastroenterology, First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, China
| | - Wen-Ge Sa
- Department of Gastroenterology, First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, China
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27
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Abstract
Hepatocellular carcinoma (HCC) is common throughout the world and most often develops as a late complication of chronic viral hepatitis or cirrhosis of any cause. As a result of the high prevalence rate of chronic hepatitis C, the incidence of HCC is rising in the United States, as well as in European and Asian countries. The overall survival rate of HCC is poor, and surgical resection and liver transplantation are the only curative treatment options. Screening for HCC offers the best hope for early detection, eligibility for treatment, and improved survival. Most physicians routinely screen at-risk patients with chronic viral hepatitis and cirrhosis for HCC, despite the lack of official guidelines. The current consensus recommendations are to screen healthy hepatitis B virus carriers with annual or semiannual serum alpha-fetoprotein; carriers with chronic hepatitis or cirrhosis and patients with cirrhosis of any etiology are surveyed with twice yearly serum alpha-fetoprotein and liver ultrasound. This article will review the current recommendations for HCC screening, the rationale that led to these recommendations, and the challenges of cost-effectiveness research in this area.
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Affiliation(s)
- Mindie H Nguyen
- Division of Gastroenterology and hepatology, Department ofMedicine, Santford, California 94304-1509, USA.
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28
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Chou SJ, Chen CM, Harn HJ, Chen CJ, Liu YC. In situ detection of hTERT mRNA relates to Ki-67 labeling index in papillary thyroid carcinoma. J Surg Res 2001; 99:75-83. [PMID: 11421607 DOI: 10.1006/jsre.2001.6124] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
BACKGROUND Telomerase is activated in most human cancers but is inactivate in adult somatic tissues except for some proliferating cell lineages. The maintenance of telomerase activity may be a critical step of cellular immortalization and transformation. MATERIALS AND METHODS We analyzed the expression of human telomerase reverse transcriptase (hTERT) using in situ hybridization and compared it to Ki-67 immunoreactivity in 29 cases of papillary thyroid carcinoma (PTC) and 17 cases of benign thyroid disease. RESULTS The hTERT messenger RNA (mRNA) was expressed in the cytoplasm of carcinoma cells with moderate (n = 10) to strong intensity (n = 10) in 69% (20 of 29) PTC cases. Human TERT was found in only 29% (5 of 17) cases of benign thyroid disease. Human TERT gene expression was preferentially detected in PTC (P = 0.021). The Ki-67 labeling index was observed in 16 cases of PTC (16 of 29; 55.2%). This result was significantly different from that of benign thyroid disease (P = 0.014). The Ki-67 labeling index related to the intensity of hTERT mRNA expression (r = 0.51; P = 0.005) and was inversely associated with the follicular variant of PTC (r = -0.413; P = 0.026). No statistically significant difference was found between hTERT expression and histological subtype of PTC. CONCLUSIONS Our results demonstrated that expression of hTERT could be detected using in situ hybridization in PTCs and was significantly distinguishable from that of benign thyroid disease. Human TERT expression was related to the Ki-67 labeling index, indicating that coupling of telomerase activation with cell proliferation was the associated mechanism for tumorigenesis.
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MESH Headings
- Adolescent
- Adult
- Aged
- Aged, 80 and over
- Antibodies, Monoclonal
- Carcinoma, Papillary/genetics
- Carcinoma, Papillary/metabolism
- Carcinoma, Papillary/pathology
- Carcinoma, Papillary, Follicular/genetics
- Carcinoma, Papillary, Follicular/metabolism
- Carcinoma, Papillary, Follicular/pathology
- DNA-Binding Proteins
- Female
- Gene Expression
- Humans
- Immunohistochemistry
- In Situ Hybridization
- Ki-67 Antigen/metabolism
- Male
- Middle Aged
- RNA
- RNA, Messenger/metabolism
- Retrospective Studies
- Telomerase/genetics
- Thyroid Diseases/genetics
- Thyroid Diseases/metabolism
- Thyroid Diseases/pathology
- Thyroid Neoplasms/genetics
- Thyroid Neoplasms/metabolism
- Thyroid Neoplasms/pathology
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Affiliation(s)
- S J Chou
- Division of General Surgery, Department of Surgery, Cardinal Tien Hospital and Fu Jen Catholic University, Taipei County, Taiwan.
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29
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Abstract
The pathogenesis of HCC is poorly understood at present. There is insufficient understanding to propose a robust general model of hepatic carcinogenesis, partly because pathogenic host and environmental factors show significant regional variation, making such generalization difficult. Figure 4 is a model based on data presented in this article. Multiple risk factors for HCC have been identified, including cirrhosis, male gender, increasing patient age, toxins, chronic viral hepatitis, and other specific liver diseases. The understanding of how the individual risk factors result in genetic changes is rudimentary, and there is even less understanding about interactions between risk factors. Future studies should acknowledge the geographic origin of the HCCs studied and consider the effects of cirrhosis, gender, and age. A more rigorous approach to these factors may help explicate the interaction with specific liver diseases so that a comprehensive model of hepatic carcinogenesis can be developed.
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Affiliation(s)
- G A Macdonald
- Queensland Institute of Medical Research and the Department of Medicine, University of Queensland, Brisbane, Queensland, Australia
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30
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Harada K, Yasoshima M, Ozaki S, Sanzen T, Nakanuma Y. PCR and in situ hybridization studies of telomerase subunits in human non-neoplastic livers. J Pathol 2001; 193:210-7. [PMID: 11180168 DOI: 10.1002/1096-9896(2000)9999:9999<::aid-path786>3.0.co;2-g] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
Telomerase, a ribonucleoprotein enzyme associated with cellular immortality, consists of human telomerase RNA component (hTERC), human telomerase protein 1 (hTEP1), and human telomerase reverse transcriptase (hTERT). In this study, the expression of these subunits was examined in non-neoplastic livers [13 cases of chronic viral hepatitis (CVH), 16 of primary biliary cirrhosis (PBC), two of primary sclerosing cholangitis, and six normal livers], using the reverse transcription-polymerase chain reaction (RT-PCR), nested PCR, and in situ hybridization (ISH). Six hepatocellular carcinoma (HCC) cases and one colonic cancer were used as positive controls. Telomeric repeat amplification protocol (TRAP) assay disclosed distinct telomerase activity in all positive controls and weak telomerase activity in non-neoplastic livers in 4 of 13 CVH cases and 5 of 16 PBC cases. By RT- and nested PCR, both hTERC and hTEP1 mRNA were detectable in all non-neoplastic liver tissues; ISH revealed hTERC and hTEP1 mRNA in the periportal and periseptal hepatocytes and inflammatory mononuclear cells in those cases examined. ISH revealed hTERT mRNA only in a few infiltrating mononuclear cells in 3 of 13 CVH and 2 of 16 PBC livers and these five cases were also positive by TRAP assay. In four of these five cases, hTERT mRNA was also detectable by nested PCR, suggesting that hTERT mRNA in the non-neoplastic liver is expressed by infiltrating mononuclear cells. Biliary epithelial cells were totally negative for these human telomerase subunits. Three subunits were constantly detected in all positive controls by ISH as well as by RT- and nested PCR. The finding that hTERC and hTEP1 mRNA, but not hTERT mRNA, were detectable in the non-neoplastic hepatocytes suggests that telomerase is present but not activated and that additional factor(s) are necessary for the expression of hTERT mRNA in the hepatocytes, along with immortalization and neoplastic transformation.
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Affiliation(s)
- K Harada
- Department of Pathology (II), Kanazawa University School of Medicine, Kanazawa, Japan
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31
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32
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Abstract
Hepatocellular carcinoma (HCC) has ranked second in cancer mortality in China since the 1990s and is increasing in frequency among males in many countries. Hepatitis B and C viruses, aflatoxin and algal toxin in the contaminated drinking water remain major aetiological factors and hepatitis G virus and transfusion-transmitted virus can not be excluded. A prospective randomized control trial screening for HCC in a high-risk population using alpha fetoprotein (AFP) and ultrasonography has demonstrated a decrease in HCC mortality. Rapidly progressing medical imaging has continuously contributed to the improving treatment results. Surgical resection still plays a major role in influencing prognosis of HCC. Studies on recurrence and metastasis after curative resection have become a key issue for further improvement of the surgical outcome. Regional cancer therapies are progressing rapidly, based on the advances in early diagnosis. The advantages and disadvantages of these are noted. Multimodality combination and sequential treatment has been accepted as an important approach for unresectable HCC and cytoreduction and sequential resection have attracted attention. Conformal radiotherapy has shown important potential for HCC treatment. Intra-arterial chemotherapy has been repeatedly proved effective; however, systemic chemotherapy for HCC remains disappointing. The effects of tamoxifen are questionable, whereas alpha-interferon has been shown to have significant potential, particularly in prevention of recurrence. All of these treatments have resulted in continuing improvement of HCC prognosis in some centres.
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Affiliation(s)
- Z Y Tang
- Liver Cancer Institute and Zhongshan Hospital, Shanghai Medical University, China.
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33
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Beerheide W, Tan YJ, Teng E, Ting AE, Jedpiyawongse A, Srivatanakul P. Downregulation of proapoptotic proteins Bax and Bcl-X(S) in p53 overexpressing hepatocellular carcinomas. Biochem Biophys Res Commun 2000; 273:54-61. [PMID: 10873563 DOI: 10.1006/bbrc.2000.2891] [Citation(s) in RCA: 45] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
As the occurrence of structural p53 mutations in hepatocellular carcinoma (HCC) in Thailand was previously reported to be much lower than that found in other high-incidence HCC areas, we analyzed 16 HCC samples from Thailand to determine the expression and functionality of p53 protein. We observed the overexpression of p53 protein in 69% of HCC, despite the prevalence of the wild-type p53 gene. However, the overexpressed p53 protein was nonfunctional as suggested by its inability to modulate the expressions of several p53 effector proteins (p21 and Bcl-2 family proteins). In addition, we observed significant underexpression of two proapoptotic proteins, Bax and Bcl-X(S), in 81% (P = 0.02) and 64% (P = 0.03) of HCC, respectively. Consequently, the ratios of proapoptotic to antiapoptotic BCL-2 family proteins were reduced in 88% of the HCC tumor tissues when compared to normal tissues, such that the rheostat between BCL-2 family proteins is strongly skewed toward enhanced cell survival in the tumor cells.
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Affiliation(s)
- W Beerheide
- Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore, 117609, Republic of Singapore
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34
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Abstract
The incidence of hepatocellular carcinoma (HCC) appears to be declining in Taiwan and potentially in other high-prevalence areas as a consequence of vaccination for hepatitis B virus (HBV). However, there is evidence that the incidence of HCC is increasing in North America and Europe. This appears to be related to the increasing prevalence and duration of hepatitis C virus (HCV) infection in these countries. There is also growing evidence to support an increase in the risk of HCC in patients with HCV who are coinfected with occult HBV (patients who have lost HBV surface antigen but still have detectable HBV DNA either in blood or liver). Occult HBV infection in patients with HCV may be more common than previously thought, and HCC that occurs in this setting appears to have a worse prognosis. There is continuing interest in the effect of interferon therapy on the incidence of HCC in patients with HCV. Several studies from Japan have shown a benefit in patients without cirrhosis, although there are a number of potentially confounding variables that may partly explain these results. Prospective randomized studies are needed to investigate this important question. The molecular biology of HCC and the events of malignant transformation in the liver continue to be areas of intense study. Recently, there has been considerable interest in telomeres, the repeat units on the ends of chromosomes, and the enzyme that maintains these, telomerase. Telomeres shorten with each cell division and can be used to determine the number of divisions a cell has undergone. Eventually they reach a critical length, with further loss resulting in cellular senescence. Telomerase restores telomere length and may help malignant cells escape senescence. Nearly all HCCs have telomerase activity and assessments of telomeres and telomerase may be clinically useful.
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Affiliation(s)
- J T Edwards
- Department of Gastroenterology and Hepatology, Royal Brisbane Hospital, Queensland Institute of Medical Research, Brisbane, Queensland, Australia
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