Evaluation Of Triethylene Tetraamine Interacting With G-quadruplexes As Telomerase Inhibitor

The reverse transcriptase telomerase is able to maintain stable telomeric length and essential for the unlimited proliferation of tumor cells. Telomerase activity has been detected in 85-90% of human tumors and tumor-derived cell lines but not in most human somatic cells. Therefore, regulation of telomerase activity could theoretically be a candidate used for anti-tumor therapy.Telomerase presents a diversity of possibilities for inhibition; one of them is to stabilize the G-quadruplexes formed by telomeric DNA. The G-rich single-stranded telomeric DNA can adopt in vitro a variety of different non-B-DNA conformations such as an intramolecular quadruplex structure (G4-DNA). A key finding has been that enzymatic activity is directly inhibited by formation of G-quadruplex structures stabilized by potassium ion. Therefore, a drug that stabilizes these tetraplexes could interfere with telomere replication, in a manner related to the effect of potassium ion on telomerase catalytic activity and processivity.In the current study, the effects of triethylene tetraamine (TETA) on the stability of G-quadruplexes, telomerase activity and the proliferation of several cell lines are explored as a potential G-quadruplex interactive agent.Circular dichroism (CD) spectroscopy is widely used to distinguish between parallel and anti-parallel G-quadruplex structures. The results, obtained from CD spectroscopy of two kinds of G-quadruplexes, indicate that TETA can accelerate the formation of parallel G-quadruplex structures formed by d(TGGGGT) and convert the intramolecular G-quadruplex structures formed by d(TTAGGG)4 from parallel to anti-parallel.From the UV melting temperature measurements, we find that TETA is able to stabilize the G-quadruplex structures with the △Tm of about 10℃ and 26℃ for d(TTAGGG)4 and d(TGGGGT) respectively at the TETA concentration of 100 μM.The effects of TETA on telomerase activity have also been examined using the modified telomeric repeat amplification protocol (TRAP) assay in cell-free extracts. The results show that TETA is a potent inhibitor of telomerase activity in the micromolar range (IC50tel = 7.8 μM). TETA also can inhibit the enzymaticactivity in HeLa cells in a concentration dependent manner. Taq polymerase assay is performed to discriminate between telomerase elongation inhibition and Taq polymerase inhibition during the amplification steps of the assay. The results demonstrate that Taq polymerase activity is not inhibited even the concentration of TETA up to 100 μM.One of the aims of anti-tumor drugs is to suppress the proliferation of tumor cells. The results of trypan blue exclusion and MTT assay show that TETA can suppress the proliferation of telomerase-positive cells including HeLa, MCF-7, K562, ECV-304, and PC12 cells (the values of IC50 of HeLa, MCF-7, K562, ECV-304, and PC12 are 75, 124, 175, 221 and 452 uM, respectively) with no acute cytotoxicity but has no effect on the HLF cell which has no telomerase activity. Cell cycle profiles of HeLa cells grown in the presence of TETA are examined by flow cytometry. The results reveal that the inhibition of telomerase activity correlates with the effects on growth arrest in the G0/G1 phase of the cell cycle in HeLa cells.In summary, TETA, a linear small molecule, has been identified as a potent telomerase inhibitor. It is able to stabilize the G-quadruplex structures and inhibit telomerase activity. Furthermore, it inhibits telomerase-positive cells proliferation and induces G0/G1 arrest for HeLa cells but without acute cytotoxicity.