Telomerase as Biomarker for Oral Cancer

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Telomerase is a ribonucleoprotein enzyme that extends telomere DNA located in the chromosomal termini. Telomerase is known to specifically express in cancer cells, and telomerase activity was found in more than 80 % of cancer patients. Therefore, telomerase may represent a promising cancer biomarker and therapeutic target. The telomerase repeat amplification protocol (TRAP) assay is a polymerase chain reaction (PCR)-based method to detect telomerase activity. In this assay, the telomerase substrate (TS) primer is elongated and its extended DNA is amplified by PCR. Telomerase activity is evaluated by a ladder of bands differing by 6 bp after gel electrophoresis of the PCR products. Recently, nonPCR-based methods to detect telomerase activity have been reported by many researchers. Electrochemical telomerase assay (ECTA), which was developed by Takenaka’s group, is a simple and rapid PCR-free method to detect S. Takenaka (*) • S. Sato Department of Applied Chemistry, Research Center for Bio-microsensing Technology, Kyushu Institute of Technology, Kitakyushu, Fukuoka, Japan e-mail: shige@che.kyutech.ac.jp; tshige@iki.bbiq.jp; shinobu@che.kyutech.ac.jp # Springer Science+Business Media Dordrecht 2015 V.R. Preedy, V.B. Patel (eds.), Biomarkers in Cancer, Biomarkers in Disease: Methods, Discoveries and Applications, DOI 10.1007/978-94-007-7681-4_8 753 telomerase activity. ECTA consists of a TS primer-immobilized electrode and ferrocenylnaphthalene diimide derivative as a tetraplex binder. Takenaka’s group compared the efficacy of ECTA and TRAP methods in detecting telomerase activity in oral cancer screening. Telomerase activity was observed in 90 % and 30 % of oral cancer tissues and exfoliative cells using the TRAP method, respectively, whereas the ECTA method detected telomerase activity in 90 % and 85 % of oral cancer tissues and exfoliative cells, respectively. These findings suggested that the ECTA method is useful for oral cancer screening because exfoliative cells can be easily obtained by scraping the inside of the mouth. Because telomerase activity is specific to cancer cells, ligands that inhibit telomerase activity may show promise as anticancer drugs. ECTA has also been used to estimate the inhibitory activity of telomerase ligands in cancer cells by determining TS primer elongation in a concentration-dependent manner. The ECTA method was used to screen for the telomerase inhibitory activity of 10 ligands and demonstrated not only the inhibitory activity of the ligands but also their mechanisms of action. The drugs having non-elongating capability directly inhibited telomerase. On the other hand, another set of drugs that can elongate at a 24-mer expansion indirectly inhibited telomerase access by binding and stabilizing the tetraplex structures. List of Abbreviations Ca9-22 Human Gingival Squamous Cell Carcinoma Cell Lines ECTA Electrochemical Telomerase Assay EtBr Ethidium Bromide FND Ferrocenylnaphthalene Diimide HSC-2 Human Oral Squamous Cell Carcinoma Cell Lines HSC-3 Human Tongue Squamous Cell Carcinoma Cell Lines hTERT Human Telomerase Reverse Transcriptase IC50 Half maximal (50 %) Inhibitory Concentration Inhibitor III Hexameric Phosphorothioate Oligonucleotide, 50-d(TTAGGG)-30 Inhibitor V 2,6-bis[3-(N-piperidino)propionamido]anthracene-9,10-dione PCR Polymerase Chain Reaction PIPER N,N0-bis [2-(1-piperidino)ethyl]-3,4,9,10-tetracarboxylic diimide Q Charge Quantity SAS Human Tongue Squamous Cell Carcinoma Cell Line SPR Surface Plasmon Resonance SWV Square Wave Voltammetry TBM 3,30,5,50-Tetramethylbenzidine TER Telomerase RNA Component TERT Telomerase Reverse Transcriptase TMPyP4 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphine TND Tri Naphthalene Diimide TRAP Telomerase Repeat Amplification protocol TS Primer Telomerase Substrate Primer 754 S. Takenaka and S. Sato