Telomere Targeting With A Novel G-quadruplex-interactive Ligand Induces Telomere Disassembly And Telomerase Displacement In Human Glioma Cells

Malignant gliomas are the most common primary tumor of the central nervous systems and represent the second leading cause of cancer-related deaths in children and young adults. The most frequent form, glioblastoma multiforme, is very aggressive and invasive and is highly refractory to anticancer treatment. Glioblastoma patients generally survive for less than 18 months after diagnosis, despite treatment by a combination of surgery, radiation therapy and chemotherapy. Although much less frequent than telomerase activation in most cancer types, it has been suggested that ALT occurs in almost 25% of glioblastoma multiforme tumorsHuman telomeres are repetitive TTAGGG sequences located on the ends of chromosomes allowing cells to distinguish between natural chromosome ends and double-strand DNA breaks. In humans, the telomere is composed of G-rich duplex with a single-stranded(ss) 3′-overhang. The 3′-overhang is either accessible for telomerase extension in an open state or inaccessible in a capped(or closed) conformation that involves the formation of loop motifs, termed as T-loop and D-loop.Uncapping of the telomere ends by different means leads to telomeric dysfunction characterized by end-to-end fusion, inappropriate recombination, anaphase bridges and G-overhang degradation that either result in apoptosis or senescence.The perpetual maintenance of telomeric DNA allows tumor cells to possess unlimited replicative potential, one of the hallmarks of cancer. Activated telomerase maintains telomere length homeostasis in ~85% of human cancers justifying the numerous anti-cancer strategies targeting components of the telomerase holoenzyme. Ligand-induced stabilization of G-quadruplex formation by the telomeric DNA single-stranded 3’-overhang inhibits telomerase from catalyzing telomeric DNA synthesis and from capping telomeric ends, making these ligands good candidates for chemotherapeutic purposes.The problem of traditional strategy for direct telomerase inhibition is the long lag period required before telomeres reach the critically short length required for eventual senescence and apoptosis. Furthermore, it has been reported that in cancer cells, inhibition of telomerase activity might activate a recombination-based alternative lengthening of telomeres mechanism for telomere maintenance, which is one of the major limitations for the development of clinically useful telomerase inhibitors.One of the most potent G-quadruplex ligands is BRACO-19, which has been shown to inhibit telomerase activity and to display antitumor activity. However, G-quadruplex-interactive molecules acting on telomeres are still in preclinical development. Therefore, identification of the underlying mechanisms of action is still a major challenge.In this report, we applied TRAP assay, immunoblotting immunofluorescence and Ch IP to analyze the effects of BRACO-19 on telomerase activity, telomere structure and cell proliferation. The main results are as follows:1. We demonstrated that BRACO-19 inhibits telomerase activity and induce selective growth suppression in glioma cells rather than normal cells, we demonstrated that BRACO-19 induces the loss of the telomerase catalytic subunit h TERT.Furthermore, Proliferation inhibition induced by BRACO-19 was associated with the DNA damage response, which was confirmed to occur at telomere.2. BRACO-19 can induce the dissociation of TRF2 and POT1(telomere binding protein)from telomere, which may provide the formation of G-quadruplex and T-loop collapse in living cells. Moreover, shorten of telomeric 3 ′-overhang exist in BRACO-19- treated glioma cells.3. The apoptosis,cell cycle arrest and senescence was as induced by BRACO-19 as consequence of telomere dysfunction. Upregulation of p53 and p21 proteins maybe associated with the growth arrest.4. BRACO-19 can induce telomere dysfunction characterized by the formation of telomere fusion and anaphase bridges. Unprotection of telomere induced by BRACO-19 visualized by the telomere-Td T assay.To our knowledge, this is the first report of BRACO-19 combining with telomere can induce viability loss in brain tumor cells. Alone, or in combination with other treatments, such as radiotherapy, they may provide a new basis for the treatment of glioblastoma multiforme, as well as other types of cancers.