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El-Desoky AM, Ali YBM, Talaat RM. Cytotoxic effect of combining two antisense oligonucleotides against telomerase rna component (hTR and mRNA of centromere protein B (CENP-B) in hepatocellular carcinoma cells. AN ACAD BRAS CIENC 2022; 94:e20200573. [PMID: 35894385 DOI: 10.1590/0001-3765202120200573] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2020] [Accepted: 07/03/2020] [Indexed: 11/22/2022] Open
Abstract
Telomerase is a ribonucleoprotein enzyme that plays a crucial role in maintaining the malignancy and is responsible for cellular immortality and tumorigenesis. On another hand, Centromere protein B (CENP-B) plays an important role in cell cycle regulation and helping in the high rate proliferation of cancer cells. Our study is designed to evaluate the effect of using combined antisense oligonucleotides (ASOs) targeting (hTR) and mRNA of CENP-B on liver cancer cells. Compared with a single treatment, combination treatment with Locked Nucleic Acid (LNA) ASO (hTR) and (CENP-B) (6.25 nM from each) exhibit the maximum synergistic cytotoxic effect. hTR and CENP-B mRNA was abrogated while hTERT expression was disappeared. Caspase-3, Bax, and Bcl-2 were not detected, indicating caspase-independent cell death. A significant reduction in [Tumor necrosis factor (TNF-α) and Transforming growth factor (TGF-β)] coincides with elevation in Nitric oxide (NO) secretions was observed. Taken together; our data suggest that combination treatment with LNA ASO (hTR) and (CENP-B) could provide a promising strategy for cancer treatment by controlling many pathways concurrently. This might open a new prospective application of antisense in cancer therapy.
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Affiliation(s)
- Ahmed M El-Desoky
- University of Sadat City (USC), Genetic Engineering and Biotechnology Research Institute (GEBRI), Department of Molecular Biology, PO Box 79 / 22857 Sadat City, Egypt
| | - Yasser B M Ali
- University of Sadat City (USC), Genetic Engineering and Biotechnology Research Institute (GEBRI), Department of Molecular Biology, PO Box 79 / 22857 Sadat City, Egypt
| | - Roba M Talaat
- University of Sadat City (USC), Genetic Engineering and Biotechnology Research Institute (GEBRI), Department of Molecular Biology, PO Box 79 / 22857 Sadat City, Egypt
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2
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Gala K, Khattar E. Long non-coding RNAs at work on telomeres: Functions and implications in cancer therapy. Cancer Lett 2021; 502:120-132. [PMID: 33450357 DOI: 10.1016/j.canlet.2020.12.036] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2020] [Revised: 12/13/2020] [Accepted: 12/29/2020] [Indexed: 12/27/2022]
Abstract
Long non-coding RNAs (lncRNAs) are known to regulate various biological processes including cancer. Cancer cells possess limitless replicative potential which is attained by telomere length maintenance while normal somatic cells have a limited lifespan because their telomeres shorten with every cell division ultimately triggering replicative senescence. Two lncRNAs have been observed to play a key role in telomere length maintenance. First is the lncRNA TERC (telomerase RNA component) which functions as a template for telomeric DNA synthesis in association with telomerase reverse transcriptase (TERT) which serves as the catalytic component. Together they constitute the telomerase complex which functions as a reverse transcriptase to elongate telomeres. Second lncRNA that helps in regulating telomere length is the telomeric repeat-containing RNA (TERRA) which is transcribed from the subtelomeric region and extends to the telomeric region. TERC and TERRA exhibit important functions in cancer with implications in precision oncology. In this review, we discuss various aspects of these important lncRNAs in humans and their role in cancer along with recent advancements in their anticancer therapeutic application.
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Affiliation(s)
- Kavita Gala
- Sunandan Divatia School of Science, SVKM's NMIMS (Deemed to be) University, Mumbai, 400056, Maharashtra, India
| | - Ekta Khattar
- Sunandan Divatia School of Science, SVKM's NMIMS (Deemed to be) University, Mumbai, 400056, Maharashtra, India.
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3
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Guittat L, Alberti P, Gomez D, De Cian A, Pennarun G, Lemarteleur T, Belmokhtar C, Paterski R, Morjani H, Trentesaux C, Mandine E, Boussin F, Mailliet P, Lacroix L, Riou JF, Mergny JL. Targeting human telomerase for cancer therapeutics. Cytotechnology 2011; 45:75-90. [PMID: 19003245 DOI: 10.1007/s10616-004-5127-z] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2004] [Accepted: 09/21/2004] [Indexed: 01/28/2023] Open
Abstract
The enzyme telomerase is involved in the replication of telomeres, specialized structures that cap and protect the ends of chromosomes. Its activity is required for maintenance of telomeres and for unlimited lifespan, a hallmark of cancer cells. Telomerase is overexpressed in the vast majority of human cancer cells and therefore represents an attractive target for therapy. Several approaches have been developed to inhibit this enzyme through the targeting of its RNA or catalytic components as well as its DNA substrate, the single-stranded 3'-telomeric overhang. Telomerase inhibitors are chemically diverse and include modified oligonucleotides as well as small diffusable molecules, both natural and synthetic. This review presents an update of recent investigations pertaining to these agents and discusses their biological properties in the context of the initial paradigm that the exposure of cancer cells to these agents should lead to progressive telomere shortening followed by a delayed growth arrest response.
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Affiliation(s)
- Lionel Guittat
- Laboratoire de Biophysique, Muséum National d'Histoire Naturelle USM503, INSERM U 565, CNRS UMR 5153, 43, rue Cuvier, 75231, Paris cedex 05, France
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4
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Xu Y. Chemistry in human telomere biology: structure, function and targeting of telomere DNA/RNA. Chem Soc Rev 2011; 40:2719-40. [DOI: 10.1039/c0cs00134a] [Citation(s) in RCA: 249] [Impact Index Per Article: 17.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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Abstract
Advances in chromosome dynamics have increased our understanding of the significant role of telomeres and telomerase in cancer. Telomerase is expressed in almost all cancer cells but is inactive in most normal somatic cells. Therefore, telomerase is an important target for the design of therapeutic agents that might have minimal side effects. Herein, we evaluate current approaches to telomerase/telomere-targeted therapy, discuss the benefits and disadvantages, and speculate on the future direction of telomerase inhibitors as cancer therapeutics.
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Abstract
Telomeres and telomerase play essential roles in the regulation of the lifespan of human cells. While normal human somatic cells do not or only transiently express telomerase and therefore shorten their telomeres with each cell division, most human cancer cells typically express high levels of telomerase and show unlimited cell proliferation. High telomerase expression allows cells to proliferate and expand long-term and therefore supports tumor growth. Owing to the high expression and its role, telomerase has become an attractive diagnostic and therapeutic cancer target. Imetelstat (GRN163L) is a potent and specific telomerase inhibitor and so far the only drug of its class in clinical trials. Here, we report on the structure and the mechanism of action of imetelstat as well as about the preclinical and clinical data and future prospects using imetelstat in cancer therapy.
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7
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Shah BA, Kaur R, Gupta P, Kumar A, Sethi VK, Andotra SS, Singh J, Saxena AK, Taneja SC. Structure-activity relationship (SAR) of parthenin analogues with pro-apoptotic activity: Development of novel anti-cancer leads. Bioorg Med Chem Lett 2009; 19:4394-8. [PMID: 19501509 DOI: 10.1016/j.bmcl.2009.05.089] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2009] [Revised: 04/28/2009] [Accepted: 05/16/2009] [Indexed: 11/26/2022]
Abstract
Analogues of parthenin were synthesized by substitutions at different reaction centres to establish a structure-activity relationship (SAR). Some of the molecules have displayed significant cytotoxicity in human cervical carcinoma (HeLa) and human myeloid leukemia (HL-60) cells. A few of the compounds also induced apoptosis in HL-60 cells measured in terms of sub-Go/G1 DNA fraction. Also one of the lead molecules has been shown to be the inhibitor of both telomerase and topoisomerase-II.
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Affiliation(s)
- Bhahwal Ali Shah
- Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu Tawi 180 001, India.
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8
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Park YP, Kim KD, Kang SH, Yoon DY, Park JW, Kim JW, Lee HG. Human telomerase reverse transcriptase (hTERT): a target molecule for the treatment of cisplatin-resistant tumors. Korean J Lab Med 2009; 28:430-7. [PMID: 19127107 DOI: 10.3343/kjlm.2008.28.6.430] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
BACKGROUND Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme that is required for telomerase activity (TA) and cancer progression. Telomerase inhibition or inactivation increases cellular sensitivity to UV irradiation, DNA-damaging agents, the tyrosine kinase inhibitor, imatinib, and pharmacological inhibitors, such as BIBR1532. hTERT is associated with apoptosis. Some patients show drug-resistance during anti-cancer drug treatment and the cancer cell acquire anti-apoptotic mechanism. Therefore, we attempted to study correlation between hTERT and drug-resistance. METHODS To study the correlation between protein level and activity of hTERT and drug-resistance, Western blotting and telomerase repeat amplification protocol (TRAP) assays were performed. To investigate whether hTERT contributes to drug resistance in tumor cells, we transiently decreased hTERT levels using small interfering RNA (siRNA) in T24/R2 cells. RESULTS hTERT knockdown increased Bax translocation into the mitochondria and cytochrome C release into the cytosol. Caspase inhibitors, especially Z-VAD-FMK, rescued this phenomenon, suggesting that the stability or expression of hTERT might be regulated by caspase activity. CONCLUSIONS These data suggest that hTERT might be a target molecule for drug-resistant tumor therapy.
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Affiliation(s)
- Yuk Pheel Park
- Medical Genomic Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea
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Bolenz C, Weiss C, Wenzel M, Gabriel U, Steidler A, Becker A, Herrmann E, Trojan L, Michel MS. In vivo evaluation of intravesical paclitaxel and combined bcl-xL antisense oligodeoxynucleotide treatment for orthotopic urothelial carcinoma. J Cancer Res Clin Oncol 2008; 135:679-86. [PMID: 18941779 DOI: 10.1007/s00432-008-0500-5] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2008] [Accepted: 10/01/2008] [Indexed: 01/01/2023]
Abstract
PURPOSE To evaluate intravesical paclitaxel monotherapy and combined treatment with antiapoptotic bcl-xL antisense oligodeoxynucleotides (AS-ODNs) on urothelial carcinoma (UC). METHODS Forty-eight FoxN(rnu) athymic nude rats with orthotopic human bladder UC were randomized to four treatment groups [1, paclitaxel; 2, paclitaxel/bcl-xL AS-ODNs; 3, bcl-xL AS-ODNs (control); 4, medium (control)]. Three consecutive instillations were applied and weekly endoscopic tumor size measurements were performed. RESULTS Significant tumor size reduction was achieved in groups 1 and 2 (each P < 0.0001), whereas continuous UC growth was observed in control animals (groups 3 and 4; P < 0.0001 and P < 0.0020). Complete tumor eradication was achieved in four treated animals (groups 1 and 2). No significant difference in chemoresection effects was found between groups 1 and 2 (P = 0.2251). CONCLUSIONS We present an in vivo evaluation of intravesical treatment with paclitaxel and combined bcl-xL AS-ODNs. Despite efficient tumor size reduction, no gain was observed when adding bcl-xL AS-ODNs in this experimental setting.
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Affiliation(s)
- Christian Bolenz
- Department of Urology, Mannheim Medical Center, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, Mannheim, Germany.
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10
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Telomerase inhibitors and 'T-oligo' as cancer therapeutics: contrasting molecular mechanisms of cytotoxicity. Anticancer Drugs 2008; 19:329-38. [PMID: 18454043 DOI: 10.1097/cad.0b013e3282f5d4c2] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Telomeres, the specialized structures that comprise the ends of chromosomes, form a closed structure, or t-loop, that is important in preventing genomic instability. Forced modulation of this structure, via overexpression of a dominant-negative form of telomere repeat binding factor 2, a protein critical for maintaining t-loop structure, for example, can result in the activation of DNA-damage responses, and ultimately cellular senescence or apoptosis. This response is also seen in normal somatic cells, where telomeres steadily decrease in length as cellular proliferation occurs owing to inefficient replication of terminal telomeric DNA. When telomere length becomes critically short, t-loop structure is compromised, and the cell undergoes senescence. Telomerase, the enzyme responsible for telomere length maintenance, is overexpressed in a majority of cancers. Its lack of expression in most normal somatic cells makes it an attractive target in designing cancer therapeutics. Compounds currently under development that seek to inhibit hTERT, the reverse transcriptase component of telomerase, include nucleoside analogs and the small molecule BIBR1532. Compounds inhibiting the RNA component of telomerase, hTERC, include peptide nucleic acids, 2-5A antisense oligonucleotides, and N3'-P5' thio-phosphoramidates. Recently, an oligonucleotide sharing sequence homology with terminal telomeric DNA, termed 'T-oligo', has shown cytotoxic effects in multiple cancers in culture and animal models. Independent of telomerase function, T-oligo is thought to mimic the DNA-damage response a cell normally experiences when the telomere t-loop structure becomes dysfunctional. In this review, the molecular mechanisms attributed to telomerase inhibitors and T-oligo, as well as their potential as cancer therapeutics, are discussed.
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11
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Phatak P, Burger AM. Telomerase and its potential for therapeutic intervention. Br J Pharmacol 2007; 152:1003-11. [PMID: 17603541 PMCID: PMC2095101 DOI: 10.1038/sj.bjp.0707374] [Citation(s) in RCA: 63] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2007] [Revised: 06/08/2007] [Accepted: 06/08/2007] [Indexed: 01/12/2023] Open
Abstract
Telomerase and telomeres are attractive targets for anticancer therapy. This is supported by the fact that the majority of human cancers express the enzyme telomerase which is essential to maintain their telomere length and thus, to ensure indefinite cell proliferation--a hallmark of cancer. Tumours have relatively shorter telomeres compared to normal cell types, opening the possibility that human cancers may be considerably more susceptible to killing by agents that inhibit telomere replication than normal cells. Advances in the understanding of the regulation of telomerase activity and the telomere structure, as well as the identification of telomerase and telomere associated binding proteins have opened new avenues for therapeutic intervention. Here, we review telomere and telomerase biology and the various approaches which have been developed to inhibit the telomere/telomerase complex over the past decade. They include inhibitors of the enzyme catalytic subunit and RNA component, agents that target telomeres, telomerase vaccines and drugs targeting binding proteins. The emerging role of telomerase in cancer stem cells and the implications for cancer therapy are also discussed.
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Affiliation(s)
- P Phatak
- Department of Pharmacology and Experimental Therapeutics; and Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine Baltimore, MD, USA
| | - A M Burger
- Department of Pharmacology and Experimental Therapeutics; and Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine Baltimore, MD, USA
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12
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De Cian A, Lacroix L, Douarre C, Temime-Smaali N, Trentesaux C, Riou JF, Mergny JL. Targeting telomeres and telomerase. Biochimie 2007; 90:131-55. [PMID: 17822826 DOI: 10.1016/j.biochi.2007.07.011] [Citation(s) in RCA: 484] [Impact Index Per Article: 26.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2007] [Accepted: 07/16/2007] [Indexed: 01/06/2023]
Abstract
Telomeres and telomerase represent, at least in theory, an extremely attractive target for cancer therapy. The objective of this review is to present the latest view on the mechanism(s) of action of telomerase inhibitors, with an emphasis on a specific class of telomere ligands called G-quadruplex ligands, and to discuss their potential use in oncology.
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Affiliation(s)
- Anne De Cian
- INSERM, U565, Acides nucléiques: dynamique, ciblage et fonctions biologiques, 43 rue Cuvier, CP26, Paris Cedex 05, F-75231, France
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13
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Abstract
The prognosis for any patient with progressive or recurrent invasive transitional cell carcinoma remains poor. In this context, the focus of clinical research in these invasive cancers concentrates on identifying systemic treatment options and new agents in order to improve survival of patients. Cisplatin-based chemotherapy is standard treatment of patients with metastatic urothelial cancer; however, despite regimens as the cisplatin-gemcitabine combination, the overall response rates vary between 40% and 65%, with complete response in 15%-25% with survivals up to 16 months. This survival is frequently achieved with severe and life-threatening side effects. None the less, almost all responding patients relapse within the first year; therefore, the need for development of new and tolerable agents is urgent. This review highlights some new active chemotherapeutic as new platinum compounds (oxaliplatin, lobaplatin), gallium nitrate, ifosfamide, the antifolates piritrexim and pemetrexed (Alimta, LY231514), vinflunine and molecular targeting agents such as farnesyltransferase inhibitors (lonafarnib, R115777, SCH66336), ribozyme (RPI.4610), histone deacetylase inhibitor (CI-994) and monoclonal antibodies (epidermal growth factor receptor, Her 2/neu).
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Affiliation(s)
- F G E Perabo
- Department of Urology, University Hospital, Bonn, Germany.
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14
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Kondo Y, Kondo S. Telomerase RNA inhibition using antisense oligonucleotide against human telomerase RNA linked to a 2',5'-oligoadenylate. Methods Mol Biol 2007; 405:97-112. [PMID: 18369820 DOI: 10.1007/978-1-60327-070-0_9] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Telomerase, a ribonucleoprotein enzyme, is detected in the vast majority of cancers, including malignant gliomas, but not in most normal somatic cells. To inhibit telomerase function effectively, we have adopted the 2',5'-oligoadenylate (2-5A) antisense system. 2-5A is a mediator of one pathway of interferon actions by activating RNase L, resulting in single-stranded RNA cleavage. By linking 2-5A to an antisense oligonucleotide, RNase L degrades the targeted RNA specifically and effectively. Therefore, we have synthesized the antisense oligonucleotide against human telomerase RNA component (hTR) linked to 2-5A (2-5A-anti-hTR) and have demonstrated its antitumor effect on telomerase-positive cancer cells in vitro and in vivo.
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Affiliation(s)
- Yasuko Kondo
- Department of Neurosurgery, Anderson Cancer Center, Houston, TX, USA
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15
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Paranjape JM, Xu D, Kushner DM, Okicki J, Lindner DJ, Cramer H, Silverman RH, Leaman DW. Human telomerase RNA degradation by 2'-5'-linked oligoadenylate antisense chimeras in a cell-free system, cultured tumor cells, and murine xenograft models. Oligonucleotides 2006; 16:225-38. [PMID: 16978086 DOI: 10.1089/oli.2006.16.225] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Ribonuclease L (RNase L) is a latent single-stranded RNA-directed endoribonuclease that is activated on binding to short 2'-5'-linked oligoadenylates (2-5A), a feature that has led to its use in antisense therapeutic strategies. By attaching a 2-5A moiety to the 5' terminus of standard antisense oligonucleotides, it is possible to activate RNase L and guide it to specific RNAs for degradation. These 2-5A antisense chimeras have been used successfully to target a variety of cellular and viral RNAs. Telomerase is a nuclear ribonucleoprotein complex that elongates telomeric DNA and contributes to cellular immortalization. Telomerase is composed of a protein catalytic subunit and an RNA (hTR or TERC) component, both of which are critical for holoenzyme activity. We describe the characterization of 2-5A antisense chimeras targeting the hTR component of telomerase (2-5A antihTR). Newly designed 2-5A anti-hTR molecules were assayed for their abilities to selectively degrade hTR in a cell-free system. Of the five chimeras tested, one (RBI011) degraded hTR by 97%, and two others (RBI013 and RBI009) were also found to be highly active (73-76% degradation). The ability of transfected RBI011, and its homolog RBI254, to degrade hTR in cultured tumor cells was assessed by real-time RT-PCR. In these studies, RBI011 and RBI254 effectively degraded hTR in a variety of hTR-positive tumor cell lines. The hTR degradation studies were extended to growth assays to determine whether hTR ablation affected tumor cell viability or proliferation. RBI254 treatment resulted in reduced tumor cell viability over the course of 4-day growth assays, effects that were augmented by cotreatment with interferon-beta. To extend these results to an in vivo system, nude mice were implanted subcutaneously or orthotopically with hTR-positive prostate tumors and treated with RBI254. RBI254-treated mice exhibited enhanced tumor cell apoptosis and reduced tumor volume as compared with controls. These findings demonstrated the effectiveness of highly active forms of 2-5A antisense against hTR, and also highlight the usefulness of the cell-free system in predicting chimera efficacy before to inception of cell-based and in vivo studies.
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Affiliation(s)
- Jayashree M Paranjape
- Department of Cancer Biology and Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA
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Synthetic nucleic acids as potential therapeutic tools for treatment of bladder carcinoma. Eur Urol 2006; 51:315-26; discussion 326-7. [PMID: 16935415 DOI: 10.1016/j.eururo.2006.07.045] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2006] [Accepted: 07/25/2006] [Indexed: 12/24/2022]
Abstract
OBJECTIVES Abnormal gene activation in human tumours including bladder cancers (bCAs) may cause altered proliferation, maturation, and apoptosis as well as development of resistance to therapeutic interventions. Therefore, silencing of abnormally activated genes appears to be a rational approach for specific target-directed and sensitising therapies. METHODS Of the available strategies for gene silencing, antisense-based techniques have attracted much attention and are the focus of this review. Putative target genes should be involved in essential tumour-promoting pathways, such as growth signalling, immortalisation, cell cycle regulation, apoptosis, angiogenesis, and development of therapy resistances. This review gives an overview of selected studies performed on bCA-derived cell lines and xenografts reporting down-regulation of potential target genes by antisense-based synthetic nucleic acids such as antisense oligodeoxynucleotides (AS-ODNs) and small interfering RNAs (siRNAs). Effects on proliferation of bCA cells and enhancement of the cytotoxic action of different chemotherapeutics were evaluated. RESULTS Knock-down of the selected target genes frequently caused an impairment of growth of different bCA cell lines originating from cell cycle arrest or increased apoptosis. In numerous studies, the pretreatment with AS-ODNs or siRNAs provoked strong enhancement of subsequent chemotherapies, emphasising the effectiveness of these inhibition approaches. CONCLUSIONS The application of antisense-based inhibitors in combination with chemotherapeutics might represent an alternative strategy for the adjuvant treatment of superficial bCA. Nevertheless, translation of this technology to the clinic might be hampered by inestimable off-target effects caused by AS-ODNs and their behaviour after intravesical instillation has to be evaluated in preclinical and clinical trials.
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17
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Fuessel S, Herrmann J, Ning S, Kotzsch M, Kraemer K, Schmidt U, Hakenberg OW, Wirth MP, Meye A. Chemosensitization of bladder cancer cells by survivin-directed antisense oligodeoxynucleotides and siRNA. Cancer Lett 2006; 232:243-54. [PMID: 16458121 DOI: 10.1016/j.canlet.2005.02.027] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2005] [Revised: 02/15/2005] [Accepted: 02/20/2005] [Indexed: 02/04/2023]
Abstract
Survivin is known to be overexpressed in numerous tumor types including human bladder cancer and to cause resistance to radiation and chemotherapy. Therefore, we tested the antisense oligodeoxynucleotide AS-SVV286 and the small interfering RNA si-SVV284 to down-regulate survivin in the BCa cell lines EJ28 and 5637 thereby acting as sensitizers for chemotherapy. Pretreatment with these inhibitors followed by chemotherapy caused an enhanced decrease in cell viability. The observed reduction in cell counts associated with increased rates of apoptosis paralleled the degree of reduction of survivin expression that was achieved more efficiently by the siRNA than by the AS-ODN. Nevertheless, both therapy approaches in combination with all tested chemotherapeutics provoked a remarkable inhibition of viability and may serve as suitable additive tools for chemosensitization of bladder cancer cells.
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Affiliation(s)
- Susanne Fuessel
- Department of Urology, Technical University Dresden, Fetscherstr. 74, D-01307 Dresden, Germany.
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18
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Glackin AJ, Gray SB, Johnston SR, Duggan BJ, Williamson KE. Antisense oligonucleotides in the treatment of bladder cancer. Expert Opin Biol Ther 2006; 5:67-77. [PMID: 15709910 DOI: 10.1517/14712598.5.1.67] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
This review examines the role that antisense oligonucleotides play in the treatment of superficial and muscle-invasive bladder cancer. The unique environment of the urinary bladder allows intravesical instillation of antisense oligonucleotides, and researchers have already demonstrated uptake of antisense oligonucleotides in models of bladder cancer. Second, proof of principle has been established by demonstrating downregulation of the antisense target mRNA and protein. Third, and most importantly from a therapeutic perspective, synergy between chemotherapy and antisense oligonucleotides has been shown in bladder cancer models in vitro and in vivo. The collective evidence points to a role for antisense oligonucleotides in the treatment of superficial and muscle-invasive bladder cancer in combination with existing treatment modalities.
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Affiliation(s)
- Anthony J Glackin
- Queen's University Belfast, Uro-oncology Group, Institute of Pathology, Grosvenor Road, Belfast, BT12 6BN, Northern Ireland.
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19
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Zhang P, Xu Q, Chen WT, Duan LQ, Zhang ZY, Zhou XJ. Synergistic down-regulation of telomerase by all-trans retinoic acid and antisense oligonucleotide in oral squamous cell carcinoma cell line (Tca8113). Oral Oncol 2006; 41:909-15. [PMID: 16051514 DOI: 10.1016/j.oraloncology.2005.05.007] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2005] [Accepted: 05/06/2005] [Indexed: 11/21/2022]
Abstract
Human telomerase, activated in about 90% of cancers, is mainly composed of hTR, hTERT and TP1. The exposed RNA template of hTR is an ideal target for antisense oligonucleotides (As-ODN); while recent findings indicate all-trans retinoid acid (ATRA) could effectively inhibit the expression of catalytic subunit-hTERT. The aim of this study was to investigate the effect of ATRA and As-ODN in oral squamous cell carcinoma and whether telomerase activity could be synergistically inhibited by them and thus therapeutically exploited in the future. As-ODN-hTR was transfected into human tongue squamous cell carcinoma cell line (Tca8113) with or without ATRA. Telomerase activity was examined by PCR-Elisa; viability was compared with growth curve; apoptotic rate was analyzed by Annexin V/PI double staining and hTERT expression was tested with western blot. Tca8113 cells displayed significant growth inhibition during the 9-day exposure to ATRA/As-ODN, especially to a combination of As-ODN-hTR and 5muM ATRA, correlating with the inhibition of telomerase expression. The relative telomerase activity was inhibited during treated with As-ODN-hTR alone, ATRA alone, or a combination of them. While without ATRA, the effect of As-ODN would disappear at 96h after transfection. As-ODN-hTR alone or combined with ATRA also significantly increase the apoptotic rate. Our findings provided direct evidence, in oral squamous cell carcinoma, As-ODN-hTR and ATRA could synergistically inhibit telomerase activity and telomerase protein in human tongue squamous cell carcinoma cells, which correlated with the induction of growth arrest.
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Affiliation(s)
- Ping Zhang
- Department of Oral and Maxillofacial Surgery, The Ninth People's Hospital, Shanghai Second Medical University, 639 Zhizaoju Road, Shanghai 200011, China
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Olaussen KA, Dubrana K, Domont J, Spano JP, Sabatier L, Soria JC. Telomeres and telomerase as targets for anticancer drug development. Crit Rev Oncol Hematol 2006; 57:191-214. [PMID: 16469501 DOI: 10.1016/j.critrevonc.2005.08.007] [Citation(s) in RCA: 63] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2005] [Revised: 08/10/2005] [Accepted: 08/11/2005] [Indexed: 12/15/2022] Open
Abstract
In most human cancers, the telomere erosion problem has been bypassed through the activation of a telomere maintenance system (usually activation of telomerase). Therefore, telomere and telomerase are attractive targets for anti-cancer therapeutic interventions. Here, we review a large panel of strategies that have been explored to date, from small inhibitors of the catalytic sub-unit of telomerase to anti-telomerase immunotherapy and gene therapy. The many positive results that are reported from anti-telomere/telomerase assays suggest a prudent optimism for a possible clinical application in a close future. However, we discuss some of the main limits for these approaches of antitumour drug development and why significant work remains before a clinically useful drug can be proposed to patients.
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Affiliation(s)
- Ken André Olaussen
- Laboratory of Radiobiology and Oncology, DSV/DRR/LRO, CEA, Fontenay aux Roses, France
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21
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Miyake H, Hara I, Fujisaw M, Gleave ME. Antisense oligodeoxynucleotide therapy for bladder cancer: recent advances and future prospects. Expert Rev Anticancer Ther 2006; 5:1001-9. [PMID: 16336091 DOI: 10.1586/14737140.5.6.1001] [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: 11/08/2022]
Abstract
Despite remarkable progress in therapeutic options for the management of bladder cancer, it remains a challenge for urologists to achieve successful outcomes in the treatment of both superficial and invasive bladder cancers. In this review, recent advances in the field of antisense oligodeoxynucleotide therapy targeting several genes playing functionally important roles in the progression and recurrence of bladder cancer are summarized. Data showing the synergistic antitumor activities of antisense oligodeoxynucleotide therapy, combined with several treatments, including cytotoxic chemotherapy, radiation and other molecular targeting therapies, are also presented. Finally, the future direction of antisense oligodeoxynucleotide therapy in the therapeutic strategy of bladder cancer is discussed. These findings may help clarify the significance of antisense oligodeoxynucleotide therapy as an attractive alternative to conventional strategies.
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Affiliation(s)
- Hideaki Miyake
- Department of Urology, Hyogo Medical Center for Adults, Akashi 673-8558, Japan.
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22
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Jiang YA, Luo HS, Fan LF, Jiang CQ, Chen WJ. Effect of antisense oligodeoxynucleotide of telomerase RNA on telomerase activity and cell apoptosis in human colon cancer. World J Gastroenterol 2004; 10:443-5. [PMID: 14760776 PMCID: PMC4724912 DOI: 10.3748/wjg.v10.i3.443] [Citation(s) in RCA: 6] [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 explore the effect of antisense oligodeoxynucleotide (As-ODN) of telomerase RNA on telomerase activity and cell apoptosis in human colon cancer.
METHODS: As-ODN was transfected into SW480 cells by liposomal transfection reagent. Telomerase activity of SW480 cells was examined by telomeric repeat amplification protocol (TRAP) and enzyme-linked immunosorbent assay (ELISA). Apoptosis was analyzed by morphology and flow cytometry.
RESULTS: The telomerase activity in SW480 cells transfected with 1.0 μmol/L of As-ODN for 2-5 days, was significantly decreased in a time-dependent manner, and the cells underwent apoptosis. The missense ODN (Ms-ODN) and the control group transfected with SW480 cells did not show these changes.
CONCLUSION: As-ODN can specifically inhibit the telomerase activity of SW480 cells and induce apoptosis.
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Affiliation(s)
- Ying-An Jiang
- Department of Gastroenterology, Renming Hospital of Wuhan University, Hubei Province, China
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23
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Du QY, Wang XB, Chen XJ, Zheng W, Wang SQ. Antitumor mechanism of antisense cantide targeting human telomerase reverse transcriptase. World J Gastroenterol 2003; 9:2030-5. [PMID: 12970900 PMCID: PMC4656668 DOI: 10.3748/wjg.v9.i9.2030] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [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 anti-tumor mechanism of antisense oligodeoxynucleotide cantide against hTERT.
METHODS: Tumor cells were cultured overnight and grown to 50%-60% confluence. HepG2 and SMMC-7721 were treated with cantide mixed with lipofectin, or lipofectin alone. After inducted for 6 h at 37 °C, 10% FCS in DMEM was replaced in each well. After the treatment repeated twice to three times in each concentration of cantide, hTERT mRNA and protein expression were measured by RT-PCR and Western blot analysis, respectively. Telomerase activity was determined by TRAP-ELISA assay. CPP32- and ICE-like activity was also investigated using CasPACE assay system at 48 h after cantide treatment, and apoptosis was evaluated using the DeadEnd assay at 24, 48 and 72 h after cantide treatment.
RESULTS: Compared to the control cells, the cells treated with cantide showed a dose-dependent decrease in hTERT mRNA levels at 24 h and in protein levels at 48 h respectively. The telomerase activity was decreased as the concentration of cantide increased at 48 h. At the concentration of 800 nM, the telomerase activity in the treated HepG2 and SMMC-7721 cells was only 17.1% (P < 0.01) and 20.3% (P < 0.01) of that in untreated cells. The levels of CPP32-like protease activity in HepG2 and SMMC-7721 increased by 2.8- and 3.0-fold (P < 0.05) at 48 h, and the levels of ICE-like protease activity also increased by 2.6- and 3.2-fold (P < 0.05) respectively. The percentage of apoptosis in HepG2 and SMMC-7721 cells treated with 800 nM cantide at 72 h was 63% and 52% (P < 0.01), respectively. By contrast, 8% and 9% of the cells were apoptosis after 72 h treatment with lipofectin alone.
CONCLUSION: Cantide can decrease telomerase activity by inhibiting the expression of hTERT gene and has a rapid anti-tumor effect through inducing the Caspase-dependent apoptosis. The rapid inhibitory effect of cantide on tumor growth demonstrates its feasibility in cancer treatment.
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Affiliation(s)
- Qing-You Du
- Beijing Institute of Radiation Med-icine, Beijing 100850, China
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24
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Rezler EM, Bearss DJ, Hurley LH. Telomere inhibition and telomere disruption as processes for drug targeting. Annu Rev Pharmacol Toxicol 2003; 43:359-79. [PMID: 12540745 DOI: 10.1146/annurev.pharmtox.43.100901.135733] [Citation(s) in RCA: 106] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
The components and cofactors of the holoenzyme telomerase and its substrate telomeric DNA are attractive targets for anticancer agents that act by inhibiting the activity of telomerase. This review outlines recent advances in telomerase inhibition that have been achieved using antisense oligonucleotides and ribozymes that target the telomerase mRNA or its hTR RNA template. Although these are potent catalytic inhibitors of telomerase, they are challenging to implement in the clinic due to their delayed effectiveness. Drugs that directly bind to the telomeres, the complex structures that are associated at the telomeric ends, and stabilize secondary DNA structures such as G-quadruplexes are also potent inhibitors of telomerase. Special focus is given here to the telomeres, the biological machinery that works in tandem with telomerase to elongate telomeres, the causes of telomere disruption or dysfunction, and the consequences of disruption/dysfunction on the activity and design of anticancer agents.
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Affiliation(s)
- Evonne M Rezler
- College of Pharmacy, The University of Arizona, Tucson, Arizona 85721, USA.
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25
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Abstract
The use of antisense (AS) oligonucleotides as therapeutic agents was proposed as far back as the 1960s/1970s when the AS strategy was initially developed. However, it has taken almost a quarter of a century for this potential to be realized. The last few years has seen a rapid increase in the number of AS molecules progressing past Phase I in clinical trials, due in part to our increased knowledge of their structure and chemistry. Here, we describe the most prominent of these modifications with respect to clinical applicability. However, the main focus of this review is clinical application, with a focus on cancer. We will discuss in detail both the status of the current AS clinical trials and the molecules that are likely to be the targets of the next group of AS molecules entering the clinic.
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Affiliation(s)
- Kathleen F Pirollo
- Department of Oncology, Georgetown University Medical Center, Washington, DC 20007, USA
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26
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Cote RJ, Datar RH. Therapeutic approaches to bladder cancer: identifying targets and mechanisms. Crit Rev Oncol Hematol 2003; 46 Suppl:S67-83. [PMID: 12850529 DOI: 10.1016/s1040-8428(03)00066-0] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022] Open
Abstract
Transitional cell carcinoma is the second most common genitourinary malignancy in US and third most common cause of death among genitourinary tumors. Treatment options for bladder cancer include surgery, often combined with chemotherapy, radiation, and/or immunotherapy. The MVAC adjuvant chemotherapy regimen has been most widely used in locally invasive as well as metastatic disease. Only a proportion of patients at risk will respond to therapy. There is thus need to identify good responder patients for adjuvant therapy and to identify new targets to treat a greater range of patients. Based upon patient-specific aberrations in pathways or known markers, both existing and new therapies can be tailored to benefit patients based on the risk of progression and molecular alterations specific to a patient's tumor. Targeted therapy, therefore, is defined as therapy that targets mechanism and risk. Utilizing the available knowledge of the molecular biology of cell-cycle regulation, signal transduction, apoptosis, and angiogenesis in bladder cancer, we review the potential therapeutic targets for rational drug development. Finally, using bladder cancer as a model for translational research, requirements for a desired clinical trial are presented.
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Affiliation(s)
- Richard J Cote
- Department of Pathology, University of Southern California Keck School of Medicine, Los Angeles, CA, USA.
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Abstract
A number of different approaches have been developed to inhibit telomerase activity in human cancer cells. Different components and types of inhibitors targeting various regulatory levels have been regarded as useful for telomerase inhibition. Most methods, however, rely on successive telomere shortening. This process is very slow and causes a long time lag between the onset of inhibition and the occurrence of senescence or apoptosis as a reversal of the immortal phenotype. Many telomerase inhibitors seem to be most efficient when combined with conventional chemotherapeutics. There are some promising approaches that seem to circumvent the slow way of telomere shortening and induce fast apoptosis in treated tumor cells. It has been demonstrated that telomerase may be involved in triggering apoptosis, but the underlying molecular mechanism remains unclear.
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28
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Ahmed A, Tollefsbol T. Telomeres, telomerase, and telomerase inhibition: clinical implications for cancer. J Am Geriatr Soc 2003; 51:116-22. [PMID: 12534855 DOI: 10.1034/j.1601-5215.2002.51019.x] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
Telomeres are located at the ends of eukaryotic chromosomes. The enzyme telomerase synthesized them, and they are responsible for maintaining the lengths of chromosomes. Absence of telomerase is associated with telomere shortening and aging of somatic cells, but high telomerase activity is observed in over 90% of human cancer cells. Although the disappearance of telomerase with aging is considered a natural defense against development of cancer, it is not known what triggers the reappearance of telomerase in cancer cells. Telomerase activity is directly correlated with the expression of its active catalytic component, the human telomerase reverse transcriptase (hTERT), which is controlled primarily at the level of transcription. An earlier paper discussed the relationship of telomerase with aging. In this article, the contemporary literature is reviewed to explore the associations between telomerase, telomerase inhibition, and cancer. Because most cancers occur in old age, with the aging of the population, the number of people suffering from cancer is expected to increase in the coming decades. It is not known what roles telomerase and hTERT play in the complex relationship between aging and cancer. Data from experimental studies suggest that telomerase assay could potentially play a role in the diagnosis and prognosis of cancers. There is also evidence that telomerase inhibitors might be used as anticancer agents. As the knowledge of the relationships between telomerase and cancer and between telomerase and aging advances, it is hoped that more about the interacting relationships between telomerase, aging, and cancer will be learned.
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Affiliation(s)
- Ali Ahmed
- Division of Gerontology and Geriatric Medicine, Department of Medicine, School of Medicine, University of Alabama at Birmingham and Heart Failure Clinic and Section of Gerontology, VA Medical Center, Birmingham, Alabama 35294-2041, USA.
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29
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Liu WJ, Jiang JF, Xiao D, Ding J. Down-regulation of telomerase activity via protein phosphatase 2A activation in salvicine-induced human leukemia HL-60 cell apoptosis. Biochem Pharmacol 2002; 64:1677-87. [PMID: 12445857 DOI: 10.1016/s0006-2952(02)01424-7] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Salvicine is a novel topoisomerase II inhibitor possessing significant antitumor activity, both in vitro and in vivo. The antitumor effect of salvicine is associated with its ability to induce tumor cell apoptosis. Telomerase plays an important role in the apoptotic pathway. However, little is known about the mechanisms of telomerase regulation during apoptosis induced by anticancer drugs. This study investigated the regulation of telomerase activity in salvicine-induced human leukemia HL-60 cell apoptosis. Salvicine treatment resulted in HL-60 cell apoptosis and down-regulation of telomerase activity in a time- and concentration-dependent manner. Repression of telomerase activity preceded a decrease in expression of the telomerase catalytic subunit (hTERT) and telomerase-associated protein (TP1) at the mRNA level, suggesting that the salvicine-induced decrease in telomerase activity may be additionally regulated by mechanisms other than telomerase subunit transcription. We observed that okadaic acid (OA), a protein phosphatase inhibitor, prevented the induction of apoptosis and the down-regulation of telomerase activity by salvicine. The significant increase in protein phosphatase 2A (PP2A) activity induced by salvicine treatment was blocked completely by OA. Moreover, although salvicine induced HL-60 cell apoptosis in a caspase-3-dependent manner, a specific caspase-3 inhibitor, Z-DEVD-FMK, did not prevent a decrease in telomerase activity or an increase in PP2A activity in apoptotic HL-60 cells, ruling out a role for caspase-3 in PP2A activation by salvicine. The results collectively suggest that the salvicine-induced decline in telomerase activity is not a consequence of HL-60 cell apoptosis and that it may be caused principally by the dephosphorylation of telomerase components mediated by PP2A activation.
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Affiliation(s)
- Wei-Jun Liu
- Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
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30
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Zaffaroni N, Lualdi S, Villa R, Bellarosa D, Cermele C, Felicetti P, Rossi C, Orlandi L, Daidone MG. Inhibition of telomerase activity by a distamycin derivative: effects on cell proliferation and induction of apoptosis in human cancer cells. Eur J Cancer 2002; 38:1792-801. [PMID: 12175697 DOI: 10.1016/s0959-8049(02)00139-9] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
In this study, we evaluated the potential of the distamycin derivative MEN 10716 as a telomerase inhibitor. Exposure of human melanoma cell extracts to MEN 10716 induced a dose-dependent inhibition of telomerase activity, with an IC50 of 24+/-3 microM. When intact JR8 melanoma cells were chronically exposed to the drug (200 microM every other day for 50 days), a marked inhibition (>80%) of the enzyme's catalytic activity was consistently observed starting from day 1. At later points in time, MEN 10716 inhibited melanoma cell proliferation and induced apoptosis. Cells surviving MEN 10716 exposure were characterised by a higher melanin content and a greater expression of p16(INK4A) protein than control cells. The effects of MEN 10716 were subsequently evaluated in different tumour cell systems. In particular, even in the H460 non-small cell lung cancer cell line, chronic exposure of the cells to the drug (100 microM every other day for 50 days) induced a consistent inhibition (>85%) of telomerase activity, a reduction of cell proliferation potential, and apoptosis. Conversely, MEN 10716 treatment did not appreciably inhibit cell proliferation in the U2-OS telomerase-negative human osteogenic sarcoma cell line. Interestingly, no variation in the mean telomere length was observed in MEN 10716-treated JR8 melanoma cells, whereas an appreciable increase in the mean telomere length was found in H460 lung cancer cells after drug exposure. Overall, the results of the study indicate that MEN 10716 is a possible telomerase inhibitor and suggest that abrogation of telomerase activity can affect cell proliferation even through pathways that are not dependent on telomere erosion.
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Affiliation(s)
- N Zaffaroni
- Dipartimento di Oncologia Sperimentale, Unità Operativa #10, Istituto Nazionale Tumori, Milan, Italy.
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31
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Feng RH, Zhu ZG, Li JF, Liu BY, Yan M, Yin HR, Lin YZ. Inhibition of human telomerase in MKN-45 cell line by antisense hTR expression vector induces cell apoptosis and growth arrest. World J Gastroenterol 2002; 8:436-40. [PMID: 12046065 PMCID: PMC4656416 DOI: 10.3748/wjg.v8.i3.436] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [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 effects of antisense human telomerase RNA (hTR) on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer.
METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made.
RESULTS: The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 h. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells.
CONCLUSION: Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.
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Affiliation(s)
- Run-Hua Feng
- Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China
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Abstract
Telomerase, a ribonucleoprotein enzyme, is considered as a potential target of cancer therapy because of its preferential expression in tumors. In particular, malignant gliomas are one of the best candidates for telomerase-targeted therapy. It is because malignant gliomas are predominantly telomerase-positive, while normal brain tissues do not express telomerase. In theory, there are two telomerase-associated therapeutic approaches for telomerase-positive tumors. One approach is the anti-telomerase cancer therapy to directly inhibit telomerase activity, resulting in apoptotic cell death or growth arrest. Two major components of the telomerase holoenzyme complex, the RNA template (hTER) and catalytic subunit (reverse transcriptase, hTERT) are well considered as therapeutic targets. The other approach is the telomerase-specific cancer therapy by targeting telomerase-expressing tumor cells as a means to directly kill the cells. Strategies using the transfer of therapeutic gene under the hTERT promoter system as well as immunotherapy directed against telomerase-positive cells are generally included. These telomerase-associated therapies are very promising for the treatment of malignant gliomas.
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Affiliation(s)
- Tadashi Komata
- The Center for Surgery Research, The Cleveland Clinic Foundation, Cleveland, Ohio, OH 44195, USA
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33
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Perry PJ, Arnold JR, Jenkins TC. Telomerase inhibitors for the treatment of cancer: the current perspective. Expert Opin Investig Drugs 2001; 10:2141-56. [PMID: 11772310 DOI: 10.1517/13543784.10.12.2141] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
Telomerase is a holoenzyme responsible for the maintenance of telomeres, the protein-nucleic acid complexes at the ends of eukaryotic chromosomes that serve to maintain chromosomal stability and integrity. Telomerase activity is essential for the sustained proliferation of most immortal cells, including cancer cells. Since the discovery that telomerase activity is detected in 85-90% of all human tumours and tumour-derived cell lines but not in most normal somatic cells, telomerase has become the focus of much attention as a novel and potentially highly-specific target for the development of new anticancer chemotherapeutics. Herein we review the current perspective for the development of telomerase inhibitors as cancer chemotherapeutics. These include antisense strategies, reverse transcriptase inhibitors and compounds capable of interacting with high-order telomeric DNA tetraplex ("G-quadruplex") structures, so as to prevent enzyme access to the necessary linear telomere substrate. Critical appraisal of each individual approach is provided together with highlighted areas of likely future development.
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Affiliation(s)
- P J Perry
- Yorkshire Cancer Research Laboratory of Drug Design, Cancer Research Group, University of Bradford, Bradford, West Yorkshire BD7 1DP, UK.
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