Inhibition Of Telomerase Activity Research Articles

Role of crocin in several cancer cell lines: An updated review.

Cancer is a major public health problem worldwide. The most important considerable features of cancer cells are uncontrolled proliferation, up-regulated differentiation, and immortality. Crocin, as a bioactive compound of saffron and as a water-soluble carotenoid has radical scavenging, anti-hyperlipidemia, memory improving, and inhibition of tumor growth effects. The present review was designed to evaluate molecular mechanisms underlying crocin effects against cancer cell lines. Data of this review have been collected from the scientific articles published in databases such as Science Direct, Scopus, PubMed, and Scientific Information Database from 1982 to 2019. According to various literature, crocin inhibits tumor growth, and its spread in several types of cancer including colorectal, pancreatic, breast, and prostate, as well as chronic myelogenous and leukemia. It inhibits telomerase activity, microtubule polymerization, cyclin D1, nuclear factor kappa B (NF-kB), multidrug resistance-associated protein (MRP1), and MRP2 overexpression. Crocin can induce apoptosis through activation of caspase 8, up-regulation of p53 expression, Bax/Bcl-2 ratio, and down-regulation expression of Bcl-2, survivin, and cyclin D1. It also down-regulates matrix metalloproteinase 2 and 9 (MMP2 and MMP9), N-cadherin, and beta-catenin expression, which are involved in tumor invasion and metastasis. Tumor invasion was also inhibited by crocin through increasing E-cadherin expression, cell cycle suppression at G1, G0/G1, S, and G2/M phases. Crocin has therapeutic and preventive effects on cancer cells line. Therefore, it has been suggested that this agent can be administered in patients that suffer from this problem.

Imetelstat Sensitizes Hematopoietic Malignancy Cells to Genotoxic Agent Via Suppression of Telomerase Mediated DNA Repair Process

Telomerase is the ribonucleoprotein enzyme that has the main function of extension of telomeric repeat. Telomerase consists of two constructions: telomerase reverse transcriptase protein (hTERT) and RNA template. Inhibition of telomerase activity is considered to be a therapeutic approach for malignant tumors since telomerase is dominantly expressed in germ cells, stem cells, and cancer cells but not in normal cells. We found that (TTAGGG)n repeats could be integrated and repair artificially induced DNA double strand break (DSB) sites (Onozawa M et al. PNAS, 2014). Therefore, we hypothesized that inhibition of telomerase activity may interfere with the DNA repair process and enhance the effects of DNA damaging agents. Imetelstat competitively suppresses telomerase activity. It has a binding site complementary to the RNA template of telomerase and induces specific inhibition. Imetelstat showed clinical benefits in patients with essential thrombocytosis or myelofibrosis used as monotherapy. However, the effectiveness of imetelstat combined with other cytotoxic agent for hematological malignancies is not clear. We therefore evaluated the synergistic anti-tumor effects of imetelstat and cytotoxic agents on hematological malignancies. First, the expression of hTERT mRNA, protein, and activity was confirmed in 8 human cell lines of hematological malignancies (U937, HL60, PALL2, CEM, IM9, RPMI8226, ST1, and SU9T01) but not in peripheral blood mononuclear cells (PBMCs) from a healthy individual. There was a significant correlation between mRNA expression and activity measured by TRAP assay (r=0.839, 95% confidence interval: 0.396-0.965, p=0.00467). The expression of hTERT mRNA in clinical samples was analyzed using primary acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) cells. The expression level of hTERT in ALL cells was significantly higher than that in AML cells (p=0.0093). The telomerase inhibitor, imetelstat had a concentration-dependent effect on telomerase inhibition, while a control oligo did not affect telomerase activity. The expression of hTERT protein was not suppressed by imetelstat treatment. Imetelstat also had a concentration-dependent suppressive effect on cell growth without affecting cell viability. Next, we evaluated the synergistic anti-tumor effects of etoposide and imetelstat at the concentration at which it did not affect cell growth by monotherapy. Cell proliferation at day 4 was inhibited by coadministration of etoposide (1 µM) and imetelstat (1 µM) compared to that with administration of etoposide alone (Figure A). Western blot analysis showed that gamma-H2AX expression level after radiation and imetelstat administration was significantly higher than that of after radiation alone (Figure B). Therefore, imetelstat enhanced DNA-DSB induced by a genotoxic agent. We determined whether the synergistic effect was mediated by shortening of telomere length. Telomere length was analyzed in long-term culture using the flow-FISH method. Imetelstat significantly shortened telomere length compared to a control oligo after day 14, while there was almost no change in telomere length at day 4. We also analyzed the changes of hTERT activity and gamma-H2AX expression after radiation in PBMCs from a healthy individual. Although hTERT activity was not detected in stable state in PBMC, hTERT activity appeared and gradually increased over time and gamma-H2AX expression peaked from 2 to 4 hours after radiation. Images of immunofluorescence staining revealed co-localization of hTERT and gamma-H2AX in the nucleus of PBMCs after radiation (Figure C). Therefore, hTERT induced by DNA damage might be directly involved in the DNA-DSB repair process. We clearly showed synergistic effects of imetelstat and a genotoxic agent in short-term treatment, in which shortening of telomeres was not observed. Potentially, telomerase inhibition could stand as a beneficial addition to other treatment methods when the inhibitor is administered at low non-toxic doses. Our hypothesis needs to be clarified in a clinical trial. Further studies are required to optimize these therapies in a variety of hematopoietic malignancies or other cancers. Figure Disclosures Nakagawa: akeda Pharmaceutical Company Limited: Research Funding. Teshima:Novartis: Honoraria, Research Funding.

Synthesis and Anticancer Activity of CDDO and CDDO-Me, Two Derivatives of Natural Triterpenoids.

Triterpenoids are natural compounds synthesized by plants through cyclization of squalene, known for their weak anti-inflammatory activity. 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), and its C28 modified derivative, methyl-ester (CDDO-Me, also known as bardoxolone methyl), are two synthetic derivatives of oleanolic acid, synthesized more than 20 years ago, in an attempt to enhance the anti-inflammatory behavior of the natural compound. These molecules have been extensively investigated for their strong ability to exert antiproliferative, antiangiogenic, and antimetastatic activities, and to induce apoptosis and differentiation in cancer cells. Here, we discuss the chemical properties of natural triterpenoids, the pathways of synthesis and the biological effects of CDDO and its derivative CDDO-Me. At nanomolar doses, CDDO and CDDO-Me have been shown to protect cells and tissues from oxidative stress by increasing the transcriptional activity of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2). At doses higher than 100 nM, CDDO and CDDO-Me are able to modulate the differentiation of a variety of cell types, both tumor cell lines or primary culture cell, while at micromolar doses these compounds exert an anticancer effect in multiple manners; by inducing extrinsic or intrinsic apoptotic pathways, or autophagic cell death, by inhibiting telomerase activity, by disrupting mitochondrial functions through Lon protease inhibition, and by blocking the deubiquitylating enzyme USP7. CDDO-Me demonstrated its efficacy as anticancer drugs in different mouse models, and versus several types of cancer. Several clinical trials have been started in humans for evaluating CDDO-Me efficacy as anticancer and anti-inflammatory drug; despite promising results, significant increase in heart failure events represented an obstacle for the clinical use of CDDO-Me.

Eighteen 5,7-Dihalo-8-quinolinol and 2,2'-Bipyridine Co(II) Complexes as a New Class of Promising Anticancer Agents.

Here we first report the design of a series of bis-chelate Co(II) 5,7-dihalo-8-quinolinol-phenanthroline derivative complexes, [Co(py)(QL1)2] (Co1), [Co(py)(QL2)2] (Co2), [Co(Phen)(QL1)2] (Co3), [Co(Phen)(QL2)2] (Co4), [Co(DPQ)(QL1)2]·(CH3OH)4 (Co5), [Co(DPQ)(QL2)2] (Co6), [Co(DPPZ)(QL1)2]·CH3OH (Co7), [Co(MDP)(QL1)2]·3H2O (Co8), [Co(ODP)(QL1)2]·CH3OH (Co9), [Co(PPT)(QL1)2]·CH3OH (Co10), [Co(ClPT)(QL1)2] (Co11), [Co(dpy)(QL3)2] (Co12), [Co(mpy)(QL1)2] (Co13), [Co(Phen)(QL4)2] (Co14), [Co(ODP)(QL4)2] (Co15), [Co(mpy)(QL4)2]I (Co16), [Co(ClPT)(QL4)2] (Co17), and [Co(ClPT)(QL5)2] (Co18), with 5,7-dihalo-8-quinolinol and 2,2′-bipyridine mixed ligands. The antitumor activity of Co1–Co18 has been evaluated against human HeLa (cervical) cancer cells in vitro (IC50 values = 0.8 nM–11.88 μM), as well as in vivo against HeLa xenograft tumor growth (TIR = 43.7%, p < 0.05). Importantly, Co7 exhibited high safety in vivo and was more effective in inhibiting HeLa tumor xenograft growth (43.7%) than cisplatin (35.2%) under the same conditions (2.0 mg/kg). In contrast, the H-QL1 and DPPZ ligands greatly enhanced the activity and selectivity of Co7 in comparison to Co1–Co6, Co8–Co18, and previously reported cobalt(II) compounds. In addition, Co7 (0.8 nM) inhibited telomerase activity, caused G2/M phase arrest, and induced mitochondrial dysfunction at a concentration 5662.5 times lower than Co1 (4.53 μM) in related assays. Taken together, Co7 showed low toxicity, and the combination could be a novel Co(II) antitumor compound candidate.

Quantifying the impact of small molecule ligands on G-quadruplex stability against Bloom helicase.

G-quadruplex (GQ) stabilizing small molecule (SM) ligands have been used to stabilize human telomeric GQ (hGQ) to inhibit telomerase activity, or non-telomeric GQs to manipulate gene expression at transcription or translation level. GQs are known to inhibit DNA replication unless destabilized by helicases, such as Bloom helicase (BLM). Even though the impact of SM ligands on thermal stability of GQs is commonly used to characterize their efficacy, how these ligands influence helicase-mediated GQ unfolding is not well understood. Three prominent SM ligands (an oxazole telomestatin derivative, pyridostatin, and PhenDC3), which thermally stabilize hGQ at different levels, were utilized in this study. How these ligands influence BLM-mediated hGQ unfolding was investigated using two independent single-molecule approaches. While the frequency of dynamic hGQ unfolding events was used as the metric in the first approach, the second approach was based on quantifying the cumulative unfolding activity as a function of time. All three SM ligands inhibited BLM activity at similar levels, 2–3 fold, in both approaches. Our observations suggest that the impact of SM ligands on GQ thermal stability is not an ideal predictor for their inhibition of helicase-mediated unfolding, which is physiologically more relevant.

Radiolabeled Oligonucleotides Targeting the RNA Subunit of Telomerase Inhibit Telomerase and Induce DNA Damage in Telomerase-Positive Cancer Cells.

Telomerase is expressed in the majority (>85%) of tumors, but has restricted expression in normal tissues. Long-term telomerase inhibition in malignant cells results in progressive telomere shortening and reduction in cell proliferation. Here we report the synthesis and characterization of radiolabeled oligonucleotides that target the RNA subunit of telomerase, hTR, simultaneously inhibiting enzymatic activity and delivering radiation intracellularly. Oligonucleotides complementary (Match) and noncomplementary (Scramble or Mismatch) to hTR were conjugated to diethylenetriaminepentaacetic dianhydride (DTPA), allowing radiolabeling with the Auger electron-emitting radionuclide indium-111 (111In). Match oligonucleotides inhibited telomerase activity with high potency, which was not observed with Scramble or Mismatch oligonucleotides. DTPA-conjugation and 111In-labeling did not change telomerase inhibition. In telomerase-positive cancer cells, unlabeled Match oligonucleotides had no effect on survival, however, 111In-labeled Match oligonucleotides significantly reduced clonogenic survival and upregulated the DNA damage marker γH2AX. Minimal radiotoxicity and DNA damage was observed in telomerase-negative cells exposed to 111In-Match oligonucleotides. Match oligonucleotides localized in close proximity to nuclear Cajal bodies in telomerase-positive cells. In comparison with Match oligonucleotides, 111In-Scramble or 111In-Mismatch oligonucleotides demonstrated reduced retention and negligible impact on cell survival. This study indicates the therapeutic activity of radiolabeled oligonucleotides that specifically target hTR through potent telomerase inhibition and DNA damage induction in telomerase-expressing cancer cells and paves the way for the development of novel oligonucleotide radiotherapeutics targeting telomerase-positive cancers. SIGNIFICANCE: These findings present a novel radiolabeled oligonucleotide for targeting telomerase-positive cancer cells that exhibits dual activity by simultaneously inhibiting telomerase and promoting radiation-induced genomic DNA damage.

Synthesis and in vitro antitumor activity evaluation of copper(II) complexes with 5-pyridin-2-yl-[1,3]dioxolo[4,5-g]isoquinoline derivatives

Seven Cu(II) complexes with 5-pyridin-2-yl-[1,3]dioxolo[4,5-g]isoquinoline derivatives as ligands: [Cu2(L1)2Cl4] (1), [Cu(L2)Cl2] (2), [Cu(L1)(NO3)2] (3), [Cu(L2)(NO3)2] (4), [Cu(L3)Cl2] (5), [Cu(L3)Br2] (6) and [Cu(L3)(NO3)2] (7){L1=9-nitro-5-pyridin-2-yl-[1,3]dioxolo[4,5-g]isoquinoline, L2=4-nitro-5-pyridin-2-yl-[1,3]dioxolo[4,5-g]isoquinoline, L3=9-bromo-5-pyridin-2-yl-[1,3]dioxolo[4,5-g]isoquinoline}, were synthesized and characterized. Their in vitro anticancer activities against T-24, MGC-80-3, HeLa, Hep-G2, A549 and SK-OV-3 were evaluated. Compared with their corresponding ligands, most of these complexes exhibited enhanced anticancer activities in contrast to their corresponding ligands and copper salt. Among them, complexes 1 and 3 displayed selective cytotoxicity to HeLa cells comparing with normal liver cell HL-7702, with IC50 values of 5.03 ± 1.20 μM and 10.05 ± 0.52 μM, respectively. Complexes 1 and 3 inhibited telomerase activity by interacting with c-myc promoter elements, and therefore exerted their antitumor activity. Furthermore, complexes 1 and 3 could trigger cell apoptosis via disruption of mitochondrial pathway through notably increased reactive oxygen species (ROS) levels, loss of mitochondrial membrane potential (Δψm), increase of the cytochrome c and apaf-1, decrease of bcl-2, and activation of caspases 3/9. Complexes 1 and 3 exhibited enhanced cytotoxicity, presenting synergetic effect after the ligands coordinated to copper(II) center.

Characterization of Telomerase (hTERT) in Solid and Hematopoietic Cancer Cell Lines Reveals Different Expression Patterns.

Overexpression of human telomerase reverse transcriptase (hTERT) allows disordered proliferation and immortality of malignant cells, which has been of interest for the development of targeted therapies. The present study aimed to characterize hTERT gene expression in a series of cancer cell lines. Leukemia cell lines K-562, its vincristine-resistant derivative K-562-Lucena1 and daunorubicin-resistant derivative FEPS; gastric adenocarcinoma lines AGP01, ACP02 and ACP03; melanoma SK-Mel-103 cells; and MN01 and MRC5, two non-neoplastic cell lines were analyzed by real-time polymerase chain reaction in order to evaluate hTERT gene expression. In leukemia cells, hTERT gene expression was significantly increased only in K-562 (p<0.05) and K-562-Lucena1 (p<0.001) when compared to the calibrator MRC5. For solid tumor types, only ACP03 presented a significant hTERT gene expression when compared to ACP02 (p<0.05). hTERT gene expression in K-562 and K-562-L ucena was significantly increased (p<0.05 to p<0.001) compared to all other cell lines except ACP03. In leukemia cell lines, hTERT gene overexpression was shown to be a potential target for pharmacological assays for drugs aiming to inhibit telomerase activity and control cell proliferation in oncohematological diseases.

BIBR1532, a Selective Telomerase Inhibitor, Enhances Radiosensitivity of Non-Small Cell Lung Cancer Through Increasing Telomere Dysfunction and ATM/CHK1 Inhibition

Telomerase is reactivated in non-small cell lung cancer (NSCLC), and it increases cell resistance to irradiation through protecting damaged telomeres and enhancing DNA damage repair. We investigated the radiosensitizing effect of BIBR1532, a highly selective telomerase inhibitor, and its corresponding mechanism in NSCLC. Cell proliferation, telomerase activity, and telomere dysfunction-induced foci were measured with CCK-8 assay, real-time fluorescent quantitative polymerase chain reaction, and immunofluorescence. The effect of BIBR1532 on the response of NSCLC cells to radiation was analyzed using clonogenic survival and xenograft tumor assays. Cell death and cell senescence induced by BIBR1532 or ionizing radiation (IR), or both, were detected with western blotting, flow cytometry, and senescence-association β-galactosidase staining assay. We observed dose-dependent direct cytotoxicity of BIBR1532 at relatively high concentrations in NSCLC cells. Low concentrations of BIBR1532 did not appear toxic to NSCLC cells; however, they substantially increased the therapeutic efficacy of IR invitro by enhancing IR-induced apoptosis, senescence, and mitotic catastrophe. Moreover, in a mouse xenograft model, BIBR1532 treatment synergized with IR at nontoxic dose levels promoted the antitumor efficacy of IR without toxicity to hematologic and internal organs. Mechanistically, lower concentrations of BIBR1532 effectively inhibited telomerase activity and increased IR-induced telomere dysfunction, resulting in disruption of chromosomal stability and inhibition of the ATM/CHK1 (ataxia-telangiectasia-mutated/Checkpoint kinase 1) pathway, which impaired DNA damage repair. Our findings demonstrate that disturbances in telomerase function by nontoxic dose levels of BIBR1532 effectively enhance the radiosensitivity of NSCLC cells. This finding provides a rationale for the clinical assessment of BIBR1532 as a radiosensitizer.

Amyloid β and Tumorigenesis: Amyloid β-Induced Telomere Dysfunction in Tumor Cells

Amyloid β and Tumorigenesis: Amyloid β-Induced Telomere Dysfunction in Tumor Cells

Quantitative Biology of Human Shelterin and Telomerase: Searching for the Weakest Point.

The repetitive telomeric DNA at chromosome ends is protected from unwanted repair by telomere-associated proteins, which form the shelterin complex in mammals. Recent works have provided new insights into the mechanisms of how human shelterin assembles and recruits telomerase to telomeres. Inhibition of telomerase activity and telomerase recruitment to chromosome ends is a promising target for anticancer therapy. Here, we summarize results of quantitative assessments and newly emerged structural information along with the status of the most promising approaches to telomerase inhibition in cancer cells. We focus on the mechanism of shelterin assembly and the mechanisms of how shelterin affects telomerase recruitment to telomeres, addressing the conceptual dilemma of how shelterin allows telomerase action and regulates other essential processes. We evaluate how the identified critical interactions of telomerase and shelterin might be elucidated in future research of new anticancer strategies.

The PinX1/NPM interaction associates with hTERT in early-S phase and facilitates telomerase activation

BackgroundTelomere maintenance is an important factor in tumorigenesis. PinX1 is a potent telomerase regulator which also involves in telomerase loading to telomeres. Nucleophosmin (NPM) can partially attenuate PinX1 inhibition of telomerase activity and NPM loading to hTERT requires PinX1. However, the role of the PinX1/NPM interaction in telomerase activity is not fully understood.MethodThe long-term effects of PinX1 and NPM down-regulation on telomere length were investigated by TRF assay. The localization of the PinX1/NPM association and the NPM/PinX1/hTERT complex formation were examined by immunofluorescence studies.ResultsConcurrent long-term down-regulation of PinX1 and NPM led to a substantial decrease in telomere length. The interaction with PinX1 was crucial in NPM localization in the nucleolus during the S phase. PinX1 and NPM associated throughout S phase and the NPM/PinX1/hTERT complex formation peaked during the early-S phase. The PinX1/NPM interaction was shown to localize away from Cajal Bodies at the start of S phase.ConclusionPinX1/NPM interaction is important in telomerase regulation during catalysis. NPM is recruited to hTERT by PinX1 and is required in the proposed telomerase modulating unit to activate telomerase when telomere extension occurs during S phase.

Stilbene Compounds Inhibit Tumor Growth by the Induction of Cellular Senescence and the Inhibition of Telomerase Activity.

Cellular senescence is a state of cell cycle arrest characterized by a distinct morphology, gene expression pattern, and secretory phenotype. It can be triggered by multiple mechanisms, including those involved in telomere shortening, the accumulation of DNA damage, epigenetic pathways, and the senescence-associated secretory phenotype (SASP), and so on. In current cancer therapy, cellular senescence has emerged as a potent tumor suppression mechanism that restrains proliferation in cells at risk for malignant transformation. Therefore, compounds that stimulate the growth inhibition effects of senescence while limiting its detrimental effects are believed to have great clinical potential. In this review article, we first review the current knowledge of the pro- and antitumorigeneic functions of senescence and summarize the key roles of telomerase in the regulation of senescence in tumors. Second, we review the current literature regarding the anticancer effects of stilbene compounds that are mediated by the targeting of telomerase and cell senescence. Finally, we provide future perspectives on the clinical utilization of stilbene compounds, especially resveratrol and pterostilbene, as novel cancer therapeutic remedies. We conclude and propose that stilbene compounds may induce senescence and may potentially be used as the therapeutic or adjuvant agents for cancers with high telomerase activity.

The Biochemical Activities of the Saccharomyces cerevisiae Pif1 Helicase Are Regulated by Its N-Terminal Domain.

Pif1 family helicases represent a highly conserved class of enzymes involved in multiple aspects of genome maintenance. Many Pif1 helicases are multi-domain proteins, but the functions of their non-helicase domains are poorly understood. Here, we characterized how the N-terminal domain (NTD) of the Saccharomyces cerevisiae Pif1 helicase affects its functions both in vivo and in vitro. Removal of the Pif1 NTD alleviated the toxicity associated with Pif1 overexpression in yeast. Biochemically, the N-terminally truncated Pif1 (Pif1ΔN) retained in vitro DNA binding, DNA unwinding, and telomerase regulation activities, but these activities differed markedly from those displayed by full-length recombinant Pif1. However, Pif1ΔN was still able to synergize with the Hrq1 helicase to inhibit telomerase activity in vitro, similar to full-length Pif1. These data impact our understanding of Pif1 helicase evolution and the roles of these enzymes in the maintenance of genome integrity.

Cyclic Naphthalene Diimide Dimer with a Strengthened Ability to Stabilize Dimeric G‐Quadruplex

A new type of dimeric cyclic naphthalene diimide derivatives (cNDI-dimers) carrying varied linker length were designed and synthesized to recognize dimeric G-quadruplex structures. All of the cNDI-dimers exhibited a high preference for recognizing G-quadruplex structures, and significantly enhanced the thermal stability of the dimeric G-quadruplex structure over the cNDI monomer by increasing the melting temperature by more than 23 °C, which indicated the strengthened ability of cNDI dimers for stabilizing dimeric G-quadruplex. cNDI dimers also showed a stronger ability to inhibit telomerase activity and stop telomere DNA elongation than cNDI monomer, which showed an improved anticancer potentiality for further therapeutic application.

Epigallocatechin-3-gallate induces telomere shortening and clastogenic damage in glioblastoma cells.

Epigallocatechingallate (EGCG) is the major polyphenol in green tea, to which many anticancer features, such as antioxidative, antigenotoxic, and antiangiogenetic properties, are attributed. Moreover, it is also well known as a telomerase inhibitor. In this work, we have chronically treated U251 glioblastoma cells with low, physiologically realistic concentrations, of EGCG, in order to investigate its effects both on telomeres and on genome integrity. Inhibition of telomerase activity caused telomere shortening, ultimately leading to senescence and telomere dysfunction at 98 days. Remarkably, we have observed DNA damage through an increase of phosphorylation of γ-H2AX histone and micronuclei also with doses and at timepoints when telomere shortening was not present. Therefore, we concluded that this DNA damage was not correlated with telomere shortening and that EGCG treatment induced not only an increase of telomere-shortening-induced senescence but also telomere-independent genotoxicity. This study questions the common knowledge about EGCG properties, but confirms the few works that indicated the clastogenic properties of this molecule, probably due to DNA reductive damage and topoisomerase II poisoning. Environ. Mol. Mutagen., 60:683-692, 2019. © 2019 Wiley Periodicals, Inc.

Insufficient PINX1 expression stimulates telomerase activation by direct inhibition of EBV LMP1-NF-κB axis during nasopharyngeal carcinoma development

ObjectiveEarly malignant transformation of nasopharyngeal carcinoma(NPC) is associated with Epstein-Barr virus(EBV) infection and telomerase activation. The EBV latent membrane protein 1(LMP1) regulates expression of various genes by triggering NF-κB signaling pathway. PINX1 is a well-identified tumor suppressor gene by inhibiting telomerase activity and cancer cell growth. However, whether and how EBV inhibit PINX1 expression and activate telomerase in NPC is still incompletely elucidated. MethodsImmunohistochemistry, real-time PCR and Western blotting were utilized to explore the expression of PINX1. Chromatin immunoprecipitation(ChIP) and Dual-luciferase reporter assay were used to elucidate the regulatory mechanism between NF-κB and PINX1. TRAP-SYBR Green assay and Southern blotting were utilized to detect telomerase activity and telomere length. CCK8 and EdU tests were conducted to measure proliferation ability. ResultsWe demonstrated that PINX1 is down-regulated in NPC for the first time. Mechanistically, we found that LMP1 could inhibit the transcriptional activity of PINX1 by promoting the binding of p65 to three specific sites in PINX1 promoter, significantly, two(-1698/-1689, tgcaatttcc; −206/-197, cgggctttac) of which have not been reported. In addition, we also observed that LMP1 overexpression resulted in increased telomerase activity, prolonged telomere length and enhanced proliferation. ConclusionWe first discovered EBV led to reduced PINX1 expression through LMP1-NF-κB-PINX1 axis, which up-regulated telomerase activity in NPC. And hence, the tumor cells acquired the ability to proliferate more exuberantly. This signaling pathway illustrates the relationship between EBV latent infection and telomerase activation, and further provides new thinking for early diagnosis and treatment in NPC.

A non-natural nucleotide uses a specific pocket to selectively inhibit telomerase activity.

Telomerase, a unique reverse transcriptase that specifically extends the ends of linear chromosomes, is up-regulated in the vast majority of cancer cells. Here, we show that an indole nucleotide analog, 5-methylcarboxyl-indolyl-2′-deoxyriboside 5′-triphosphate (5-MeCITP), functions as an inhibitor of telomerase activity. The crystal structure of 5-MeCITP bound to the Tribolium castaneum telomerase reverse transcriptase reveals an atypical interaction, in which the nucleobase is flipped in the active site. In this orientation, the methoxy group of 5-MeCITP extends out of the canonical active site to interact with a telomerase-specific hydrophobic pocket formed by motifs 1 and 2 in the fingers domain and T-motif in the RNA-binding domain of the telomerase reverse transcriptase. In vitro data show that 5-MeCITP inhibits telomerase with a similar potency as the clinically administered nucleoside analog reverse transcriptase inhibitor azidothymidine (AZT). In addition, cell-based studies show that treatment with the cell-permeable nucleoside counterpart of 5-MeCITP leads to telomere shortening in telomerase-positive cancer cells, while resulting in significantly lower cytotoxic effects in telomerase-negative cell lines when compared with AZT treatment.

Effects of MCRS1 on proliferation, migration, invasion, and epithelial mesenchymal transition of gastric cancer cells by interacting with Pkmyt1 protein kinase

Microspherule protein 1(MCRS1) is known to be an oncogene in several tumors. However, recent studies have shown that MCRS1 inhibits lymphatic metastasis in gastric cancer (GC) patients by inhibiting telomerase activity. Protein kinase, membrane associated tyrosine/threonine 1(Pkmyt1), a member of the WEE1 family, has been found to interact with MCRS1 by yeast two-hybrid assay; however, how these two proteins interact in GC is still unclear. Hence, this study aimed to investigate the effect of MCRS1 interaction with Pkmyt1 on GC cell proliferation, migration, and invasion. Initially, we observed increased expression of MCRS1 in GC SGC-7901 cells and decreased expression in GC BGC-823 cells. Hence, we down-regulated MCRS1 expression in SGC-7901 cells and up-regulated it in BGC-823 cells. Our results showed that overexpression of MCRS1 inhibits the growth, invasion and migration of GC cells, while downregulation of MCRS1 promotes the growth, invasion and migration of GC cells. When MK1775, an inhibitor of WEE1 kinase, was added after downregulation of MCRS1, phenotypic recovery effects were observed. Overexpression of MCRS1 also inhibited the expression of Pkmyt1 and vice versa. This indicated that there might be a possible interaction between MCRS1 and Pkmyt1. Furthermore, immunoprecipitation assay revealed the interaction between MCRS1 and Pkmyt1 in virto, and immunofluorescence experiments showed that the two proteins were co-localized in the cytoplasm. In conclusion, our study confirmed the specific tumor suppressive activity of MCRS1 in GC proliferation, invasion and migration and suggested that it might inhibit the progression of GC through its interaction with Pkmyt1.

A novel antisense oligonucleotide anchored on the intronic splicing enhancer of hTERT pre-mRNA inhibits telomerase activity and induces apoptosis in glioma cells

Alternative splicing of hTERT pre-mRNA is an important step in the regulation of telomerase activity, but the regulation mechanisms and functions remain unclear. RT-PCR analysis was used to detect hTERT splicing in glioma cell lines and brain tissues. TRAP assay was used to detect the telomerase activity. Then, we designed and synthesized 2'-O-methyl-RNA phosphorothioate AONs and transfected them into glioma cells to detect the changes in telomerase activity. MTT assay, plate colony formation assay, western blotting and Annexin V/PI assay were used to detect cell proliferation and apoptosis. At last, bioinformatics analyses were used to predict the expression and function of splicing protein SRSF2 in gliomas. hTERT splicing occurs both in glioma cell lines and glioma patients' tissues. The telomerase activity was related to the expression level of the full-length hTERT, rather than the total hTERT transcript level. AON-Ex726 was complementary to the sequence of the intronic splicing enhancer (ISE) in intron six, and significantly altered the splicing pattern of hTERT pre-mRNA, reducing the expression level of the full-length hTERT mRNA and increasing the expression level of the -β hTERT mRNA. After transfection with AON-Ex726, the level of apoptosis was increased, while telomerase activity and cell proliferation were significantly decreased. By bioinformatic predictions, we found the AON-Ex726 anchoring sequence in ISE overlaps the binding site of SRSF2 protein, which is up-regulated during the development of gliomas. Our findings provided new targets and important clues for the gene therapy of gliomas by regulating the alternative splicing pattern of hTERT pre-mRNA.