Tomentosin Induces Telomere Shortening and Caspase-Dependant Apoptosis in Cervical Cancer Cells.

Tomentosin, a natural sesquiterpene lactone purified from of Inula viscosa L., was investigated for its anti-proliferative, telomere shortening, and apoptotic effects on human cervical cancer HeLa and SiHa cell lines. Tomentosin was found to inhibit the growth of SiHa and HeLa cell lines in dose and time-dependent manner (IC50 values of 7.10 ± 0.78 µM and 5.87 ± 0.36 µM, respectively after 96 h of treatment). As evidenced by TTAGGG telomere length assay, tomentosin target specifically the telomeric overhang lengthening. This was confirmed by the evaluation of the cytotoxic effects of tomentosin in the foetal fibroblast Wi38 and JW10 cells which were derived from Wi38 and express hTERT, the telomerase catalytic subunit. We found that JW10 cells are 4.7-fold more sensitive to tomentosin which argues for telomere as its specific target. Furthermore, we found that tomentosin mediate this cytotoxic effect by inducing apoptosis and cell cycle arrest at G2/M phase. Morphological features of treated cells, as evidenced by Hoechst 33324 staining, revealed that the cytotoxic effect was due to induction of apoptosis. This was accompanied by pro-caspase-3 cleavage, an increase in caspase-3 activity and a cleavage of poly (ADP-ribose) polymerase (PARP). Moreover, tomentosin induced a decrease in mitochondrial membrane potential (ΔΨm) and an increase in reactive oxygen species (ROS), accompanied by a decrease in Bcl-2 expression. This indicates that tomentosin-induced apoptosis may involve a mitochondria-mediated signaling pathway. This study provides the first evidence that tomentosin targets telomere machinery and induces apoptosis in cervical cancer cells. The molecular mechanism underlying tomentosin-induced apoptosis may involve a mitochondria-mediated signaling pathway. J. Cell. Biochem. 118: 1689-1698, 2017. © 2016 Wiley Periodicals, Inc.

Inula Viscosa Extracts Induces Telomere Shortening and Apoptosis in Cancer Cells and Overcome Drug Resistance.

Telomerase is activated in human papillomavirus (HPV) positive cervical cancer and targeting telomeres offers a novel anticancer therapeutic strategy. In this study, the telomere targeting properties, the cytotoxic as well as the pro-apoptotic effects of hexane (IV-HE) and dichloromethane (IV-DF) fractions from Inula viscosa L. extracts were investigated on human cervical HeLa and SiHa cancer cells. Our data demonstrate that IV-HE and IV-DF extracts were able to inhibit cell growth in HeLa and SiHa cells in a dose-dependent manner and studied resistant cell lines exhibited a resistance factor less than 2 when treated with the extracts. IV-HE and IV-DF extracts were able to inhibit telomerase activity and to induce telomere shortening as shown by telomeric repeat amplification protocol and TTAGGG telomere length assay, respectively. The sensitivity of fibroblasts to the extracts was increased when telomerase was expressed. Finally, IV-HE and IV-DF were able to induce apoptosis as evidenced by an increase in annexin-V labeling and caspase-3 activity. This study provides the first evidence that the IV-HE and IV-DF extracts from Inula viscosa L. target telomeres induce apoptosis and overcome drug resistance in tumor cells. Future studies will focus on the identification of the molecules involved in the anticancer activity.

DNA damage signaling induced by the G-quadruplex ligand 12459 is modulated by PPM1D/WIP1 phosphatase.

The triazine derivative 12459 is a potent G-quadruplex ligand that triggers apoptosis or delayed growth arrest, telomere shortening and G-overhang degradation, as a function of its concentration and time exposure to the cells. We have investigated here the DNA damage response induced by 12459 in A549 cells. Submicromolar concentrations of 12459 triggers a delayed Chk1-ATR-mediated DNA damage response associated with a telomeric dysfunction and a G2/M arrest. Surprisingly, increasing concentrations of 12459 leading to cell apoptosis induced a mechanism that bypasses the DNA damage signaling and leads to the dephosphorylation of Chk1 and γ-H2AX. We identified the phosphatase Protein Phosphatase Magnesium dependent 1D/Wild-type P53-Induced Phosphatase (PPM1D/WIP1) as a factor responsible for this dephosphorylation. SiRNA-mediated depletion of PPM1D/WIP1 reactivates the DNA damage signaling by 12459. In addition, PPM1D/WIP1 is activated by reactive oxygen species (ROS) induced by 12459. ROS generated by 12459 are sufficient to trigger an early DNA damage in A549 cells when PPM1D/WIP1 is depleted. However, ROS inactivation by N-acetyl cysteine (NAC) treatment does not change the apoptotic response induced by 12459. Because PPM1D expression was recently reported to modulate the recruitment of DNA repair molecules, our data would suggest a cycle of futile protection against 12459, thus leading to a delayed mechanism of cell death.

Effects of a halogenated G-quadruplex ligand from the pyridine dicarboxamide series on the terminal sequence of XpYp telomere in HT1080 cells.

Non-canonical four-stranded structures called G-quadruplexes can form among telomere repeats during its replication. Small molecule ligands able to interact and to stabilize G-quadruplexes were shown to disrupt the binding of essential telomeric components, such as POT1 and to trigger a telomeric dysfunction associated with a delayed growth arrest in tumor cells. We describe here the chemical synthesis and the G-quadruplex binding properties of three halogenated analogs of the 360A ligand that belongs to the 2,6 pyridine dicarboxamide series. 360A is now commonly used as a benchmark both for biophysical and cellular assays as this compound was shown to display a potent affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA and to induce delayed growth inhibition in HT1080 tumor cell line. Two biophysical assays indicate that, in most cases, the presence of the halogen atom seems to slightly improve the interaction with the telomeric quadruplex. For stability reasons, the bromo derivative (360A-Br) was selected for the cellular assays. Since POT1 participates to the fine tuning of the C-strand end resection during telomere replication, we investigated the effect of 360A-Br to alter the terminal nucleotide composition of XpYp telomere in HT1080 cells using C-STELA. HT1080 cells treated for up to 24 days with 360A-Br presented some minor but significant variations of C-strand terminal nucleotide composition, also observed with a partial siRNA depletion of POT1. The relevance of these minor modifications of the telomeric C-strand resection induced by 360A-Br in HT1080 cells are discussed.

The G-quadruplex ligand telomestatin impairs binding of topoisomerase IIIalpha to G-quadruplex-forming oligonucleotides and uncaps telomeres in ALT cells.

In Alternative Lengthening of Telomeres (ALT) cell lines, specific nuclear bodies called APBs (ALT-associated PML bodies) concentrate telomeric DNA, shelterin components and recombination factors associated with telomere recombination. Topoisomerase IIIalpha (Topo III) is an essential telomeric-associated factor in ALT cells. We show here that the binding of Topo III to telomeric G-overhang is modulated by G-quadruplex formation. Topo III binding to G-quadruplex-forming oligonucleotides was strongly inhibited by telomestatin, a potent and specific G-quadruplex ligand. In ALT cells, telomestatin treatment resulted in the depletion of the Topo III/BLM/TRF2 complex and the disruption of APBs and led to the segregation of PML, shelterin components and Topo III. Interestingly, a DNA damage response was observed at telomeres in telomestatin-treated cells. These data indicate the importance of G-quadruplex stabilization during telomere maintenance in ALT cells. The function of TRF2/Topo III/BLM in the resolution of replication intermediates at telomeres is discussed.

A novel small molecule that alters shelterin integrity and triggers a DNA-damage response at telomeres.

We describe a novel synthetic small molecule which shows an unprecedented stabilization of the human telomeric G-quadruplex with high selectivity relative to double-stranded DNA. We report that this compound can be used in vitro to inhibit telomerase activity and to uncap human POT1 (protection of telomeres 1) from the telomeric G-overhang. We also show that the small molecule G-quadruplex binder induces a partial alteration of shelterin through POT1 uncapping from telomeres in human HT1080 cancer cells and the presence of gammaH2AX foci colocalized at telomeres.

DNA and RNA quadruplex ligands.

Guanine-rich nucleic acids can adopt unusual structures called guanine quadruplexes (G4) based on stacked guanine quartets. Both RNA and DNA backbones are compatible with G4 formation. As RNA and DNA quadruplexes may be recognized by ligands, it is important to understand the rules that govern the stability and specificity of these complexes. We explore the binding of a pyridine dicarboxamide derivative to various oligoribo- and oligodeoxyribo-nucleotides.

Targeting telomeres and telomerase.

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.

A new steroid derivative stabilizes g-quadruplexes and induces telomere uncapping in human tumor cells.

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG) with a 3' single-stranded extension (the G-overhang). The stabilization of G-quadruplexes in the human telomeric sequence by small-molecule ligands inhibits the activity of telomerase and results in telomere uncapping, leading to senescence or apoptosis of tumor cells. Therefore, the search for new and selective G-quadruplex ligands is of considerable interest because a selective ligand might provide a telomere-targeted therapeutic approach to treatment of cancer. We have screened a bank of derivatives from natural and synthetic origin using a temperature fluorescence assay and have identified two related compounds that induce G-quadruplex stabilization: malouetine and steroid FG. These steroid derivatives have nonplanar and nonaromatic structures, different from currently known G-quadruplex ligands. Malouetine is a natural product isolated from the leaves of Malouetia bequaaertiana E. Woodson and is known for its curarizing and DNA-binding properties. Steroid FG, a funtumine derivative substituted with a guanylhydrazone moiety, interacted selectively with the telomeric G-quadruplex in vitro. This derivative induced senescence and telomere shortening of HT1080 tumor cells at submicromolar concentrations, corresponding to the phenotypic inactivation of telomerase activity. In addition, steroid FG induced a rapid degradation of the telomeric G-overhang and the formation of anaphase bridges, characteristics of telomere uncapping. Finally, the expression of protection of telomere 1 (POT1) induced resistance to the growth effect of steroid FG. These results indicate that these steroid ligands represent a new class of telomere-targeted agents with potential as antitumor drugs.

Differential modulation of nuclear texture, histone acetylation, and MDR1 gene expression in human drug-sensitive and -resistant OV1 cell lines.

Image cytometric study of pathological specimens or cell lines has suggested that epigenetic mechanisms are likely to play a major role in determining chromatin patterns evaluable through nuclear texture analysis. We previously reported that nuclear textural changes observed in the OV1-VCR etoposide-resistant ovarian carcinoma cell line were associated with an increased acetylated histone H4 level. In this study we analyzed the effects of treatments with the HDAC inhibitor trichostatin A (TSA) or with nickel subsulfide on histone H4 acetylation, nuclear texture, and MDR1 gene expression in drug-sensitive IGROV1 and drug-resistant OV1-VCR cell lines. In IGROV1 cells, TSA induced an increase in acetylated H4 level associated with a chromatin textural decondensation and an increase in MDR1 gene expression. In OV1-VCR cells, a similar increase in H4 acetylation was observed, but nuclear texture or MDR1 gene expression remained unchanged. ChIP analysis revealed that MDR1 gene expression remained stable in TSA-treated OV1-VCR cells despite a localized increase in H4 acetylation at the promoter level. Analysis of the methylation status of MDR1 promoter showed an increase in DNA methylation at 3 specific sites in OV1-VCR cells, that could participate to TSA low responsiveness in these cells. Treatment with nickel subsulfide induced a decrease in H4 acetylation without any effect on nuclear texture characteristics in both cell lines. In OV1-VCR cells, nickel subsulfide induced a significant down-regulation of the MDR1 gene expression. These results indicate that modulation of histone H4 acetylation level can be associated with up- or down-regulation of the MDR1 gene in OV1 cells. However, this modulation does not always result in chromatin pattern alterations and these data emphasize the complexity of chromatin texture regulation in tumor cells.

Telomestatin-induced telomere uncapping is modulated by POT1 through G-overhang extension in HT1080 human tumor cells.

Telomestatin is a potent G-quadruplex ligand that interacts with the 3' telomeric overhang, leading to its degradation, and induces a delayed senescence and apoptosis of cancer cells. POT1 and TRF2 were recently identified as specific telomere-binding proteins involved in telomere capping and t-loop maintenance and whose interaction with telomeres is modulated by telomestatin. We show here that the treatment of HT1080 human tumor cells by telomestatin induces a rapid decrease of the telomeric G-overhang and of the double-stranded telomeric repeats. Telomestatin treatment also provokes a strong decrease of POT1 and TRF2 from their telomere sites, suggesting that the ligand triggers the uncapping of the telomere ends. The effect of the ligand is associated with an increase of the gamma-H2AX foci, one part of them colocalizing at telomeres, thus indicating the occurrence of a DNA damage response at the telomere, but also the presence of additional DNA targets for telomestatin. Interestingly, the expression of GFP-POT1 in HT1080 cells increases both telomere and G-overhang length. As compared with HT1080 cells, HT1080GFP-POT1 cells presented a resistance to telomestatin treatment characterized by a protection to the telomestatin-induced growth inhibition and the G-overhang shortening. This protection is related to the initial G-overhang length rather than to its degradation rate and is overcome by increased telomestatin concentration. Altogether these results suggest that telomestatin induced a telomere dysfunction in which G-overhang length and POT1 level are important factors but also suggest the presence of additional DNA sites of action for the ligand.

Downregulation by a long-acting beta2-adrenergic receptor agonist and corticosteroid of Staphylococcus aureus-induced airway epithelial inflammatory mediator production.

Although Staphylococcus aureus is a major cause of pulmonary infection, the role played by this bacterium in the induction of inflammation of human airway epithelial cells (HAEC) is poorly understood. In this study, we investigated the inflammatory response of HAEC to S. aureus soluble virulence factors and demonstrate that the combination of a long-acting beta2-adrenergic receptor agonist (salmeterol) with a glucocorticoid (fluticasone propionate) has an anti-inflammatory effect on HAEC. First, we demonstrate increased expression at both the mRNA and protein levels of interleukin (IL)-8, IL-6, and tumor necrosis factor (TNF)-alpha following incubation of HAEC in the presence of S. aureus soluble virulence factors and the increase of 1) the free nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) activities and 2) the phosphorylated (P-) extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2), the P-c-Jun NH2-terminal kinase (JNK), and the P-isoform-alpha of the NF-kappaB inhibitor (IkappaB alpha). Next, when HAEC were preincubated with the combination of salmeterol and fluticasone propionate, the inflammatory response of HAEC was markedly attenuated in that levels of IL-8, IL-6, TNF-alpha, NF-kappaB, AP-1, P-ERK1/ERK2, P-JNK, and P-IkappaB alpha decreased significantly. These data emphasize the deleterious effect of S. aureus soluble virulence factors and suggest that the combination of a beta2-adrenergic receptor agonist with a glucocorticoid may attenuate the associated airway epithelial inflammation.

Transcriptional and post-transcriptional regulation of glutathione S-transferase P1 expression during butyric acid-induced differentiation of K562 cells.

Over-expression of glutathione S-transferase P1 is related to chemotherapeutic drug resistance as well as to differentiation of human erythroleukemia cells. In opposition to previously described differentiating inducers which enhance the GST-resistance phenotype, time- and concentration-dependent activation of both erythroid and megakaryocytic differentiation pathways by butyric acid progressively diminished GSTP1 mRNA expression. GSTP1 mRNA expression decreased by 25% (p<0.01) and 64% (p<0.01) in 1mM and 2mM butyric acid-differentiated K562 cells, respectively. These results were associated to both a reduction of GATA-1 binding activity to the GSTP1 promoter and to a posttranscriptional destabilization of GSTP1 mRNA in a concentration dependent manner. Indeed, GSTP1 mRNA half-life decreased from 43.8 to 36.2 h and 12.6 h in 1mM- and 2mM-treated cells, respectively.

The benzophenanthridine alkaloid fagaronine induces erythroleukemic cell differentiation by gene activation.

Fagaronine, a benzophenanthridine alkaloid from Fagara zanthoxyloides Lam. (Rutaceae), has been tested on the erythroleukemic cell line K562 in order to explain some previous results on cell differentiation. In this study we showed that fagaronine induces a significant hemoglobinization of the human erythroleukemic cell line K562. This hemoglobin synthesis was accompanied by a strong increase of erythroid mRNA expression such as gamma- and alpha-globin, and PBGD, an enzyme of heme synthesis. In addition, the Epo-R transcripts were also stimulated indicating that cells are engaged in a maturation process. Both transcription factors GATA-1 and NF-E2, which play an important role in the regulation of genes involved in the erythroid differentiation, were also transcriptionally up-regulated. To elucidate the possible role of GATA-1 in the FAG-induced differentiation of K562 cells, we transfected reporter constructs containing regulatory regions of erythroid genes encompassing GATA-1 binding sites. After 48 hours of treatment, FAG stimulated the EPO-R and gamma-globin promoters by 2- to 3-fold and the promoter/enhancer region of GATA-1 gene by 3.2-fold. A mutation within the GATA-1 binding sites strongly decreased the promoter activation induced by FAG. Taken together, our results represent a demonstration that FAG exerts its differentiating activity by a specific activation of the regulating GATA-1 regions of genes involved in the erythroid phenotype expression.

[Regulation of telomeres length: making the telomeres accessible?]. / Régulation de la longueur des télomères: rendre le télomère accessible?

Under a normal state, the extremities of chromosomes, telomeres, are protected against undesired fusion events. Alterations of the telomere structure are associated with genetic instability, while erosion of the telomeric repeats, occurring at each cell division, provides a mechanism controlling the long-term proliferation of somatic cells. Although the structure and composition of the human telomerase enzyme are now well characterized, the protein partners regulating the stability and conformation of its DNA substrate, the telomeric end, are much less known. A functional link has been recently evidenced between proteins that bind the double-stranded telomere repeats and those recruited at the 3' G-rich telomeric overhang. This review presents an update on these telomeric factors controlling telomere lengthening and discuss the actual models proposed for its regulation.

Overexpression of Bcl-2 is associated with apoptotic resistance to the G-quadruplex ligand 12459 but is not sufficient to confer resistance to long-term senescence.

The triazine derivative 12459 is a potent G-quadruplex interacting agent that inhibits telomerase activity. This agent induces time- and dose-dependent telomere shortening, senescence-like growth arrest and apoptosis in the human A549 tumour cell line. We show here that 12459 induces a delayed apoptosis that activates the mitochondrial pathway. A549 cell lines selected for resistance to 12459 and previously characterized for an altered hTERT expression also showed Bcl-2 overexpression. Transfection of Bcl-2 into A549 cells induced a resistance to the short-term apoptotic effect triggered by 12459, suggesting that Bcl-2 is an important determinant for the activity of 12459. In sharp contrast, the Bcl-2 overexpression was not sufficient to confer resistance to the senescence-like growth arrest induced by prolonged treatment with 12459. We also show that 12459 provokes a rapid degradation of the telomeric G-overhang in conditions that paralleled the apoptosis induction. In contrast, the G-overhang degradation was not observed when apoptosis was induced by camptothecin. Bcl-2 overexpression did not modify the G-overhang degradation, suggesting that this event is an early process uncoupled from the final apoptotic pathway.

Increased glutathione S-transferase P1-1 expression by mRNA stabilization in hemin-induced differentiation of K562 cells.

GSTP1-1 gene expression mechanisms were investigated in hemin-induced erythroid differentiation of K562 cells. Hemoglobin production during differentiation was followed by a significant increase in GSTP1-1 mRNA (1.7-fold, P < 0.01) and protein (1.2-fold, P < 0.01) after 4 days of induction. This increase in mRNA production was not due to transcriptional up-regulation by GATA-1 previously shown to regulate GSTP1-1 during erythroid and megakaryocytic differentiation. Moreover, a drastic decrease in differentiation-specific GATA-1 mRNA expression was correlated to a reduction in GATA-1 promoter binding activity. Neither AP-1 nor NF-kappaB transcription factor binding activities could provide an explanation to the GSTP1-1 mRNA overexpression in hemin-treated cells. GSTP1-1 mRNA stability analysis using actinomycin D as an inhibitor of mRNA neosynthesis showed that mRNA half-life was doubled in hemin-induced erythroid differentiation of K562 cells. These results allow us to add stabilization of GSTP1-1 mRNA as a novel regulatory mechanism during hemin-mediated differentiation of K562 cells.

Targeting human telomerase for cancer therapeutics.

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.

Expression of glutathione S-transferase P1-1 in differentiating K562: role of GATA-1.

Glutathione S-transferase P1-1 (GSTP1-1) conjugates glutathione to electrophilic compounds and its expression is correlated to chemotherapeutic drug resistance. Results show that GSTP1-1 mRNA as well as protein expressions are increased during Aclarubicin (Acla)- and Doxorubicin (Dox)-induced erythroid differentiation of human K562 cells. In contrast, during megakaryocytic differentiation by 12-O-tetradecanoyl phorbol 13-acetate (TPA), GSTP1-1 expression decreased at both mRNA and protein levels. In order to clarify the molecular mechanisms leading to these variations, we identified a GATA sequence located at -1208 relative to the transcriptional start site of the GSTP1-1 promoter. By gel shift, competition, and supershift analyses we show here the specificity of the GATA-1 binding regulated by both anthracyclines and TPA. Altogether, these results demonstrate for the first time the implication of GATA-1 in differentiation-specific variations of GSTP1-1 expression.

Resistance to the short term antiproliferative activity of the G-quadruplex ligand 12459 is associated with telomerase overexpression and telomere capping alteration.

Ligands that stabilize the telomeric G-rich single-stranded DNA overhang into G-quadruplex can be considered as potential antitumor agents that block telomere replication. Ligand 12459, a potent G-quadruplex ligand that belongs to the triazine series, has been previously shown to induce both telomere shortening and apoptosis in the human A549 cell line as a function of its concentration and time exposure. We show here that A549 clones obtained after mutagenesis and selected for resistance to the short term effect of ligand 12459 frequently displayed hTERT transcript overexpression (2-6-fold). Overexpression of hTERT was also characterized in two resistant clones (JFD10 and JFD18) as an increase in telomerase activity, leading to an increase in telomere length. An increased frequency of anaphase bridges was also detected in JFD10 and JFD18, suggesting an alteration of telomere capping functions. Transfection of either hTERT or DN-hTERT cDNAs into A549 cells did not confer resistance or hypersensitivity to the short term effect of ligand 12459, indicating that telomerase expression is not the main determinant of the antiproliferative effect of ligand 12459. In contrast, transfection of DN-hTERT cDNA into resistant JFD18 cells restored sensitivity to apoptotic concentrations of ligand 12459, suggesting that telomerase does participate in the resistance to this G-quadruplex ligand. This work provides evidence that telomerase activity is not the main target for the 12459 G-quadruplex ligand but that hTERT functions contribute to the resistance phenotype to this class of agents.