IMPDH Inhibition Decreases TERT Expression and Synergizes the Cytotoxic Effect of Chemotherapeutic Agents in Glioblastoma Cells.

IMP dehydrogenase (IMPDH) inhibition has emerged as a new target therapy for glioblastoma multiforme (GBM), which remains one of the most refractory tumors to date. TCGA analyses revealed distinct expression profiles of IMPDH isoenzymes in various subtypes of GBM and low-grade glioma (LGG). To dissect the mechanism(s) underlying the anti-tumor effect of IMPDH inhibition in adult GBM, we investigated how mycophenolic acid (MPA, an IMPDH inhibitor) treatment affected key oncogenic drivers in glioblastoma cells. Our results showed that MPA decreased the expression of telomerase reverse transcriptase (TERT) in both U87 and U251 cells, and the expression of O6-methylguanine-DNA methyltransferase (MGMT) in U251 cells. In support, MPA treatment reduced the amount of telomere repeats in U87 and U251 cells. TERT downregulation by MPA was associated with a significant decrease in c-Myc (a TERT transcription activator) in U87 but not U251 cells, and a dose-dependent increase in p53 and CCCTC-binding factor (CTCF) (TERT repressors) in both U87 and U251 cells. In U251 cells, MPA displayed strong cytotoxic synergy with BCNU and moderate synergy with irinotecan, oxaliplatin, paclitaxel, or temozolomide (TMZ). In U87 cells, MPA displayed strong cytotoxic synergy with all except TMZ, acting primarily through the apoptotic pathway. Our work expands the mechanistic potential of IMPDH inhibition to TERT/telomere regulation and reveals a synthetic lethality between MPA and anti-GBM drugs.

Anti-Leukemic Effects of Small Molecule Inhibitor of c-Myc (10058-F4) on Chronic Myeloid Leukemia Cells.

BACKGROUND: As one of the main molecules in BCR-ABL signaling, c-Myc acts as a pivotal key in disease progression and disruption of long-term remission in patients with CML. OBJECTIVES: To clarify the effects of c-Myc inhibition in CML, we examined the anti-tumor property of a well-known small molecule inhibitor of c-Myc 10058-F4 on K562 cell line. METHODS: This experimental study was conducted in K562 cell line for evaluation of cytotoxic activity of 10058-F4 using Trypan blue and MTT assays. Flow cytometry and Quantitative RT-PCR analysis were also conducted to determine its mechanism of action. Additionally, Annexin/PI staining was performed for apoptosis assessment. RESULTS: The results of Trypan blue and MTT assay demonstrated that inhibition of c-Myc, as shown by suppression of c-Myc expression and its associated genes PP2A, CIP2A, and hTERT, could decrease viability and metabolic activity of K562 cells, respectively. Moreover, a robust elevation in cell population in G1-phase coupled with up-regulation of p21 and p27 expression shows that 10058-F4 could hamper cell proliferation, at least partly, through induction of G1 arrest. Accordingly, we found that 10058-F4 induced apoptosis via increasing Bax and Bad; In contrast, no significant alterations were observed NF-KB pathway-targeted anti-apoptotic genes in the mRNA levels. Notably, disruption of the NF-κB pathway with bortezomib as a common proteasome inhibitor sensitized K562 cells to the cytotoxic effect of 10058-F4, substantiating the fact that the NF-κB axis functions probably attenuate the K562 cells sensitivity to c-Myc inhibition. CONCLUSIONS: It can be concluded from the results of this study that inhibition of c-Myc induces anti-neoplastic effects on CML-derived K562 cells as well as increases the efficacy of imatinib. For further insight into the safety and effectiveness of 10058-F4 in CML, in vivo studies will be required.

Targeting Telomere Dynamics as an Effective Approach for the Development of Cancer Therapeutics.

Telomere is a protective structure located at the end of chromosomes of eukaryotes, involved in maintaining the integrity and stability of the genome. Telomeres play an essential role in cancer progression; accordingly, targeting telomere dynamics emerges as an effective approach for the development of cancer therapeutics. Targeting telomere dynamics may work through multifaceted molecular mechanisms; those include the activation of anti-telomerase immune responses, shortening of telomere lengths, induction of telomere dysfunction and constitution of telomerase-responsive drug release systems. In this review, we summarize a wide variety of telomere dynamics-targeted agents in preclinical studies and clinical trials, and reveal their promising therapeutic potential in cancer therapy. As shown, telomere dynamics-active agents are effective as anti-cancer chemotherapeutics and immunotherapeutics. Notably, these agents may display efficacy against cancer stem cells, reducing cancer stem levels. Furthermore, these agents can be integrated with the capability of tumor-specific drug delivery by the constitution of related nanoparticles, antibody drug conjugates and HSA-based drugs.

Therapeutic targeting of PLK1 in TERT promoter-mutant hepatocellular carcinoma.

BACKGROUND: Hotspot mutations in the promoter of telomerase reverse transcriptase (TERT) gene are the most common genetic variants in hepatocellular carcinoma (HCC) and associated with poor prognosis of the disease. However, no drug was currently approved for treating TERT promoter mutation positive HCC patients. Here, we aim to explore the potential therapeutic strategy for targeting TERT promoter mutation in HCC. METHODS: The Liver Cancer Model Repository database was used for screening potential drugs to selectively suppress the growth of TERT promoter mutant HCC cells. RNA-seq, CRISPR-Cas9 technology and siRNA transfection were performed for mechanistic studies. Cell counting kit-8 (CCK8) assay and the xenograft tumour models were used for cell growth detection in vitro and in vivo, respectively. Cell apoptosis and cell cycle arrest were analysed by Annexin V-FITC staining and/or propidium iodide staining. RESULTS: PLK1 inhibitors were remarkably more sensitive to HCC cells harbouring TERT promoter mutation than wild-type cells in vitro and in vivo, which were diminished after TERT promoter mutation was edited to the wild-type nucleotide. Comparing the HCC cells with wild-type promoter of TERT, PLK1 inhibitors specifically downregulated Smad3 to regulate TERT for inducing apoptosis and G2/M arrest in TERT mutant HCC cells. Moreover, knockout of Smad3 counteracted the effects of PLK1 inhibitors in TERT mutant HCC cells. Finally, a cooperative effect of PLK1 and Smad3 inhibition was observed in TERT mutant cells. CONCLUSIONS: PLK1 inhibition selectively suppressed the growth of TERT mutant HCC cells through Smad3, thus contributed to discover a novel therapeutic strategy to treat HCC patients harbouring TERT promoter mutations. KEY POINTS: TERT promoter mutation confers sensitivity to PLK1 inhibitors in HCC. The selective growth inhibition of TERT mutant HCC cells induced by PLK1 inhibitor was mediated by Smad3. Combined inhibition of PLK1 and Smad3 showed a cooperative anti-tumor effect in TERT mutant HCC cells.

Therapeutic targeting of telomerase ameliorates experimental choroidal neovascularization.

Choroidal neovascularization (CNV) is the principal driver of blindness in neovascular age-related macular degeneration (nvAMD). Increased activity of telomerase, has been associated with endothelial cell proliferation, survival, migration, and invasion in the context of tumor angiogenesis. Expanding on this knowledge, we investigated the role of telomerase in the development of CNV in mouse model. We observed increased gene expression and activity of telomerase in mouse CNV. Genetic deficiency of the telomerase components, telomerase reverse transcriptase (Tert) and telomerase RNA component (Terc) suppressed laser-induced CNV in mice. Similarly, a small molecule inhibitor of TERT (BIBR 1532), and antisense oligonucleotides (ASOs) targeting Tert and Terc reduced CNV growth. Bone marrow chimera studies suggested that telomerase activity in non-bone marrow-derived cells is crucial for the development of CNV. Comparison of BIBR 1532 with VEGF neutralizing therapeutic strategy in mouse revealed a comparable level of angiosuppressive activity. However, when BIBR and anti-VEGF antibodies were administered as a combination at sub-therapeutic doses, a statistically significant suppression of CNV was observed. These findings underscore the potential benefits of combining sub-therapeutic doses of BIBR and anti-VEGF antibodies for developing newer therapeutic strategies for NV-AMD. Telomerase inhibition with BIBR 1532 suppressed induction of multiple cytokines and growth factors critical for neovascularization. In conclusion, our study identifies telomerase as a promising therapeutic target for treating neovascular disease of the eye and thus provides a proof of principle for further exploration of telomerase inhibition as a novel treatment strategy for nvAMD.

Evaluation of the anticancer effect of telomerase inhibitor BIBR1532 in anaplastic thyroid cancer in terms of apoptosis, migration and cell cycle.

Anaplastic thyroid cancer (ATC) represents the type with the worst prognosis among thyroid cancers. In ATC with a highly invasive phenotype, selective targeting of TERT with BIBR1532 may be a goal-driven approach to preserving healthy tissues. In present study, it was aimed to investigate the effects of treatment of SW1736 cells with BIBR1532 on apoptosis, cell cycle progression, and migration. The apoptotic effect of BIBR1532 on SW1736 cells was examined using the Annexin V method, the cytostatic effect using cell cycle test, migration properties using wound healing assay. Gene expression differences were determined by real-time qRT-PCR and differences in protein level by ELISA test. BIBR1532-treated SW1736 cells had 3.1-fold increase in apoptosis compared to their untreated counterpart. There was 58.1% arrest in the G0/G1 phase and 27.6% arrest in the S phase of the cell cycle in untreated group, treatment with BIBR1532 increased cell population in G0/G1 phase to 80.9% and decreased in S phase to 7.1%. Treatment with the TERT inhibitor resulted in a 50.8% decrease in cell migration compared to the untreated group. After BIBR1532 treatment of SW1736 cells, upregulation of BAD, BAX, CASP8, CYCS, TNFSF10, CDKN2A genes, and downregulation of BCL2L11, XIAP, CCND2 genes were detected. BIBR1532 treatment resulted in an increase in BAX and p16 proteins, and a decrease in concentration of BCL-2 protein compared to untreated group. Targeting TERT with BIBR1532 as a mono drug or using of BIBR1532 at "priming stage" prior to chemotherapy treatment in ATC may present a novel and promising treatment strategy.

Therapeutic cancer vaccination against telomerase: clinical developments in melanoma.

PURPOSE OF REVIEW: Checkpoint inhibitors (CPIs) have revolutionized treatment outcomes for patients with malignant melanoma. Long-term follow-up shows that a substantial subset of patients who exhibit clinical responses achieve extended overall survival. Nevertheless, most patients do not achieve durable benefit from CPIs, and improvements are urgently needed. The clinical efficacy of CPIs depends on highly variable preexisting spontaneous T-cell immune responses. Cancer vaccines represent an independent treatment modality uniquely capable of expanding the repertoire of tumor-specific T cells in cancer patients and thus have the capacity to compensate for the variability in spontaneous T-cell responses. Vaccines are, therefore, considered attractive components in a CPI-combination strategy. RECENT FINDINGS: Here we discuss recent results obtained through therapeutic vaccination against telomerase human telomerase reverse transcriptase (hTERT). Recent publications on translational research and clinical results from phase I trials indicate that vaccination against telomerase in combination with CPIs provides relevant immune responses, negligible added toxicity, and signals of clinical efficacy. CONCLUSION: In the near future, randomized data from clinical trials involving therapeutic cancer vaccines and checkpoint inhibitors will be available. Positive readout may spark broad development and allow cancer vaccines to find their place in the clinic as an important component in multiple future CPI combinations.

Identification of mechanism of cancer-cell-specific reactivation of hTERT offers therapeutic opportunities for blocking telomerase specifically in human colorectal cancer.

Transcriptional reactivation of hTERT is the limiting step in tumorigenesis. While mutations in hTERT promoter present in 19% of cancers are recognized as key drivers of hTERT reactivation, mechanisms by which wildtype hTERT (WT-hTERT) promoter is reactivated, in majority of human cancers, remain unknown. Using primary colorectal cancers (CRC) we identified Tert INTeracting region 2 (T-INT2), the critical chromatin region essential for reactivating WT-hTERT promoter in CRCs. Elevated ß-catenin and JunD level in CRC facilitates chromatin interaction between hTERT promoter and T-INT2 that is necessary to turn on hTERTexpression. Pharmacological screens uncovered salinomycin, which inhibits JunD mediated hTERT-T-INT2 interaction that is required for the formation of a stable transcription complex on the hTERT promoter. Our results showed for the first time how known CRC alterations, such as APC, lead to WT-hTERT promoter reactivation during stepwise-tumorigenesis and provide a new perspective for developing cancer-specific drugs.

Healthy and cancer cells harbor the same DNA sequence, but reactivation of the Human Telomerase Reverse Transcriptase (hTERT) gene is observed only in cancer cells. How does that happen was not known for over three decades of research? This study identifies a specific DNA structure that forms only in cancer cells and brings the necessary molecular machinery into the correct position to activate the hTERT gene. The detailed mechanism of hTERT activation provided in this study will be instrumental in designing cancer cell-specific hTERT inhibitors, especially since all the other ways of inhibiting telomerase failed in the clinic.

A combination of telomerase inhibition and NK cell therapy increased breast cancer cell line apoptosis.

Triple-negative breast cancer (TNBC) is a subtype of breast tumor with the highest breast cancer stem cells (BCSCs) content and resistance to conventional treatment. Due to the immunosuppressive tumor microenvironment and immunogenicity of breast cancer cells, the use of immune cells, especially natural killer cells (NK) in the treatment of solid tumors, including breast cancer, has been unsatisfactory. Therefore, identifying novel therapies is requisite for breast cancer treatment. Furthermore, the combination of cancer therapies is an effective strategy to improve therapeutic effectiveness. In this study, we inhibited telomerase (hTERT) with BIBR1532, in stimulating NK cell cytotoxicity against breast cancer cells. The MDA-MB-231 cell line was cured with IC50 level of BIBR1532 for 24 h. Afterward, cells were washed with PBS and were co-cultured with peripheral blood NK cell for 5h. Finally, we assessed the impact of telomerase inhibition on the cytotoxicity of NK cells and apoptosis of breast cancer. Also, the expression of hTERT and apoptotic-related genes were evaluated. The data revealed that inhibition of telomerase increases NK cell cytotoxicity against breast cancer. Furthermore, telomerase inhibition and NK cell synergistically enhanced cell death in breast cancer cells by suppressing hTERT, upregulation of bax, and bad expression. In conclusion, telomerase suppression makes breast cancer cells more sensitive to NK cell therapy. Consequently, the combination of telomerase inhibition and NK cells can be useful in the treatment of breast cancer cells.

Targeting telomerase reverse transcriptase with the covalent inhibitor NU-1 confers immunogenic radiation sensitization.

Beyond synthesizing telomere repeats, the telomerase reverse transcriptase (TERT) also serves multiple other roles supporting cancer growth. Blocking telomerase to drive telomere erosion appears impractical, but TERT's non-canonical activities have yet to be fully explored as cancer targets. Here, we used an irreversible TERT inhibitor, NU-1, to examine impacts on resistance to conventional cancer therapies. In vitro, inhibiting TERT sensitized cells to chemotherapy and radiation. NU-1 delayed repair of double-strand breaks, resulting in persistent DNA damage signaling and cellular senescence. Although NU-1 alone did not impact growth of syngeneic CT26 tumors in BALB/c mice, it dramatically enhanced the effects of radiation, leading to immune-dependent tumor elimination. Tumors displayed persistent DNA damage, suppressed proliferation, and increased activated immune infiltrate. Our studies confirm TERT's role in limiting genotoxic effects of conventional therapy but also implicate TERT as a determinant of immune evasion and therapy resistance.

Evaluation of the efficacy of MST-312, as a telomerase inhibitor, in the treatment of patients with multiple myeloma after stem cell transplantation.

High-dose chemotherapy and stem cell transplantation are the best treatment options in patients with multiple myeloma. Numerous medicines have been studied as a maintenance treatment after transplantation. Still, the use of medications that, in addition to their maintenance properties, eliminate or delay relapse of the disease has always been researchers' purpose. Therefore, this study was performed to evaluate the efficacy of MST-312 after stem cell transplantation in patients with multiple myeloma. For this purpose, 73 patients with multiple myeloma after stem cell transplantation were studied. Thirty-five patients were in the case group, and 37 patients were in the control group. The case group was treated with 100 mg/day MST-312. Stem cell survival was evaluated in the two groups. Also, the expression of TNFα and IL-6 genes were evaluated by the Real-time PCR technique. The results showed no significant difference between the two groups in terms of stem cell survival in the first year (P=0.72) and second years of treatment (P=0.66). But there was a significant difference between the two groups regarding progression-free survival (PFS) in the first year (P=0.041) and the second year (P=0.029). These results indicate that MST-312 inhibits the progress of the disease by inhibiting the telomerase activity of myeloma cells. Genetic evaluations also showed that IL-6 and TNF-α genes were significantly reduced in the case group. Therefore, it could be suggested that MST-312 has a selective inhibitory effect on myeloma cell growth and can be indicated as a suitable candidate for treating multiple myeloma.

Telomerase inhibition on acute myeloid leukemia stem cell induced apoptosis with both intrinsic and extrinsic pathways.

AIMS: Acute Myeloid Leukemia (AML) is an invasive and lethal blood cancer caused by a rare population of Leukemia Stem Cells (LSCs). Telomerase activation is a limitless self-renewal process in LSCs. Apart from telomerase role in telomere lengthening, telomerase (especially hTERT subunit) inhibits intrinsic-, extrinsic-, and p53- mediated apoptosis pathways. In this study, the effect of Telomerase Inhibition (TI) on intrinsic-, extrinsic-, p53-mediated apoptosis, and DNMT3a and TET epigenetic markers in stem (CD34+) and differentiated (CD34-) AML cells is evaluated. MAIN METHODS: High-purity CD34+ (primary AML and KG-1a) cells were enriched using the Magnetic-Activated Cell Sorting (MACS) system. CD34+ and CD34- (primary AML and KG-1a) cells were treated with BIBR1532 and then, MTT assay, Annexin V/7AAD, Ki-67 assay, Telomere Length (TL) measurement, and transcriptional alterations of p53, hTERT, TET2, DNMT3a were analyzed. Finally, apoptosis-related genes and proteins were studied. KEY FINDINGS: TI with the IC50 values of 83.5, 33.2, 54.3, and 24.6 µM in CD34+ and CD34- (primary AML and KG-1a) cells significantly inhibited cell proliferation and induced apoptosis. However, TI had no significant effect on TL. The results also suggested TI induced intrinsic-, extrinsic-, and p53-mediated apoptosis. It was shown that the expression levels of DNMT3a and TET2 epigenetic markers were highly increased following TI. SIGNIFICANCE: In total, it was revealed that TI induced apoptosis through intrinsic, extrinsic, and p53 pathways and increased the expression of DNMT3a and TET2 epigenetic markers.

Anti-Proliferative and Anti-Telomerase Effects of Blackberry Juice and Berry- Derived Polyphenols on HepG2 Liver Cancer Cells and Normal Human Blood Mononuclear Cells.

BACKGROUND: Previous studies have provided strong evidence for the anticancer activity of berry fruits. OBJECTIVE: In this study, we investigated the effects of blackberry juice and three berry- polyphenolic compounds on cell proliferation and telomerase activity in human hepatoma HepG2 and normal peripheral blood mononuclear cells (PBMCs). METHODS: The cell viability and telomerase activity were measured by MTT and TRAP assay, respectively. Berry effects on the expression of genes were determined by quantitative RT-PCR assay. RESULTS: Blackberry, gallic acid, and resveratrol inhibited proliferation of both HepG2 and PBMC cells in a dosedependent manner. Resveratrol was more effective than gallic acid for reducing the viability of HepG2 cells, but both showed the same level of growth inhibition in PBMC cells. Berry, resveratrol, and gallic acid significantly inhibited telomerase activity in HepG2 cells. The antiproliferative effect of berry was associated with apoptotic DNA fragmentation. Gallic acid was more effective for reducing telomerase activity than resveratrol, but anthocyanin moderately increased telomerase activity in cancer cells. Telomerase activity was induced by all three polyphenols in PBMCs. Overall, Krumanin chloride was more effective to induce telomerase than gallic acid and resveratrol in PBMC cells. There was no significant difference in hTERT, hTR, and Dnmts expressions between berry treated and the control untreated HepG2 cells. But, a significant downregulation of HDAC1 and HDAC2 and upregulation of SIRT1 were observed in berry-treated cells. CONCLUSION: These data indicate that the berry anticancer effect is associated with antitelomerase activity and changes in HDACs expression. The data also suggest that berry antitelomerase activity is mainly related to its gallic acid and resveratrol, but not anthocyanin content.

Shikonin N-benzyl matrinic acid ester derivatives as novel telomerase inhibitors with potent activity against lung cancer cell lines.

In this study, a series of novel shikonin N-benzyl matrinic acid ester derivatives (PMMB-299-PMMB-310) were synthesized and tested for their ability to inhibit the proliferation of cancer cells. Compared with shikonin and matrine, some of the ester derivatives were found to exhibit better anti-proliferative activity against seven different cancer cell lines, with less cytotoxicity toward non-cancerous cells. The strongest anti-proliferative activity was exhibited by PMMB-302, which had an IC50 value of 2.71 µM against A549 cells. The compound caused cell cycle arrest in the G2/M phase and induced apoptosis. Effects on the expression of apoptosis-related molecules such as Bcl2, Bcl-XL, caspase-3, caspase-9 and FADD suggested that PMMB-302 has tumor suppressive roles in lung cancer cells. In addition, PMMB-302 inhibited expression of telomerase core proteins, dyskerin and NHP2, and telomerase reverse transcriptase RNA. Moreover, molecular docking of PMMB-302 was subsequently conducted to determine the probable binding mode with telomerase. Taken together, the results indicate that PMMB-302 acts as a tumor suppressor in lung cancer cells by negatively regulating telomerase expression.

Research on Radiosensitivity of the Protein Kinase B Signaling Pathway in Cervical Cancer.

The main characteristics of cervical cancer are abnormal and uncontrolled cell proliferation, and it regulates cell growth, differentiation, and cell death through genetic and epigenetic changes. This paper mainly discusses the radiosensitivity of the cervical cancer protein kinase B signaling pathway and discusses the specific mechanisms that affect the occurrence and development of cervical cancer. In addition, this paper studies the effect of transient transfection knocking down the expression of TRIP4 in cervical cancer cells on the expression of key proteins in related signaling pathways and explores the mechanism of its specific effects and finds the mechanism of TRIP4's effect on cervical cancer radiosensitivity. The findings of this study show for the first time that knocking down TRIP4 inhibits cell viability by inhibiting the P13K/AKT and MAPK/ERK pathways, and this corresponds to the first part of the experimental results, which show that knocking down TRIP4 inhibits colony formation and increases apoptosis in HeLa and SiHa cells. Moreover, simultaneous inhibition of TRIP4 and hTERT proteins can increase the radiosensitivity of cervical cancer cells. These findings indicate that the inhibition of TRIP4 may be a new type of treatment that selectively targets the P13K/AKT and MAPK/ERK pathways and hTERT pathways in cervical cancer cells and provides a therapeutic option for the treatment of cervical cancer.

Effects of in vitro short- and long-term treatment with telomerase inhibitor in U-251 glioma cells.

BACKGROUND: The inhibition of the enzyme telomerase (TERT) has been widely investigated as a new pharmacological approach for cancer treatment, but its real potential and the biochemical consequences are not totally understood. OBJECTIVE: Here, we investigated the effects of the telomerase inhibitor MST-312 on a human glioma cell line after both short- and long-term (290 days) treatments. METHODS: Effects on cell growth, viability, cell cycle, morphology, cell death and genes expression were assessed. RESULTS: We found that short-term treatment promoted cell cycle arrest followed by apoptosis. Importantly, cells with telomerase knock-down revealed that the toxic effects of MST-312 are partially TERT dependent. In contrast, although the long-term treatment decreased cell proliferation at first, it also caused adaptations potentially related to treatment resistance and tumor aggressiveness after long time of exposition. CONCLUSIONS: Despite the short-term effects of telomerase inhibition not being due to telomere erosion, they are at least partially related to the enzyme inhibition, which may represent an important strategy to pave the way for tumor growth control, especially through modulation of the non-canonical functions of telomerase. On the other hand, long-term exposure to the inhibitor had the potential to induce cell adaptations with possible negative clinical implications.

Zinc protoporphyrin binding to telomerase complexes and inhibition of telomerase activity.

Zinc protoporphyrin (ZnPP), a naturally occurring metalloprotoporphyrin (MPP), is currently under development as a chemotherapeutic agent although its mechanism is unclear. When tested against other MPPs, ZnPP was the most effective DNA synthesis and cellular proliferation inhibitor while promoting apoptosis in telomerase positive but not telomerase negative cells. Concurrently, ZnPP down-regulated telomerase expression and was the best overall inhibitor of telomerase activity in intact cells and cellular extracts with IC50 and EC50  values of ca 2.5 and 6 µM, respectively. The natural fluorescence properties of ZnPP enabled direct imaging in cellular fractions using non-denaturing agarose gel electrophoresis, western blots, and confocal fluorescence microscopy. ZnPP localized to large cellular complexes (>600 kD) that contained telomerase and dysskerin as confirmed with immunocomplex mobility shift, immunoprecipitation, and immunoblot analyses. Confocal fluorescence studies showed that ZnPP co-localized with telomerase reverse transcriptase (TERT) and telomeres in the nucleus of synchronized S-phase cells. ZnPP also co-localized with TERT in the perinuclear regions of log phase cells but did not co-localize with telomeres on the ends of metaphase chromosomes, a site known to be devoid of telomerase complexes. Overall, these results suggest that ZnPP does not bind to telomeric sequences per se, but alternatively, interacts with other structural components of the telomerase complex to inhibit telomerase activity. In conclusion, ZnPP actively interferes with telomerase activity in neoplastic cells, thus promoting pro-apoptotic and anti-proliferative properties. These data support further development of natural or synthetic protoporphyrins for use as chemotherapeutic agents to augment current treatment protocols for neoplastic disease.

The Interactions of H2TMPyP, Analogues and Its Metal Complexes with DNA G-Quadruplexes-An Overview.

The evidence that telomerase is overexpressed in almost 90% of human cancers justifies the proposal of this enzyme as a potential target for anticancer drug design. The inhibition of telomerase by quadruplex stabilizing ligands is being considered a useful approach in anticancer drug design proposals. Several aromatic ligands, including porphyrins, were exploited for telomerase inhibition by adduct formation with G-Quadruplex (GQ). 5,10,15,20-Tetrakis(N-methyl-4-pyridinium)porphyrin (H2TMPyP) is one of the most studied porphyrins in this field, and although reported as presenting high affinity to GQ, its poor selectivity for GQ over duplex structures is recognized. To increase the desired selectivity, porphyrin modifications either at the peripheral positions or at the inner core through the coordination with different metals have been handled. Herein, studies involving the interactions of TMPyP and analogs with different DNA sequences able to form GQ and duplex structures using different experimental conditions and approaches are reviewed. Some considerations concerning the structural diversity and recognition modes of G-quadruplexes will be presented first to facilitate the comprehension of the studies reviewed. Additionally, considering the diversity of experimental conditions reported, we decided to complement this review with a screening where the behavior of H2TMPyP and of some of the reviewed metal complexes were evaluated under the same experimental conditions and using the same DNA sequences. In this comparison under unified conditions, we also evaluated, for the first time, the behavior of the AgII complex of H2TMPyP. In general, all derivatives showed good affinity for GQ DNA structures with binding constants in the range of 106-107 M-1 and ligand-GQ stoichiometric ratios of 3:1 and 4:1. A promising pattern of selectivity was also identified for the new AgII derivative.

Antisense oligonucleotide repress telomerase activity via manipulating alternative splicing or translation.

Telomerase is a reverse transcriptase that catalyzes the addition of telomeric repeated DNA onto the 3' ends of linear chromosomes. Telomerase inhibition was broadly used for cancer therapeutics. Here, six antisense oligonucleotides were designed to regulate TERT mRNA alternative splicing and protein translation. To pursue a better stability in vitro, we chemically modified the oligonucleotides into phosphorothioate (PS) backbone and 2'-O-methoxyethyl (2'-MOE PS) version and phosphoroamidate morpholino oligomer (PMO) version. The oligonucleotides were transfected into HEK 293T cells and HeLa cells, and the mRNA expression, protein level and catalytic activity of telomerase were determined. We found the Int8 notably promoted hTERT mRNA exon 7-8 skipping, which greatly reduced telomerase activity, and the 5'-UTR treatment led to an obvious protein translation barrier and telomerase inhibition. These results demonstrate the potential of antisense oligonucleotide drugs targeting hTERT for antitumor therapy. Moreover, two specific antisense oligonucleotides were identified to be effective in reducing telomerase activity.

[The Effect and Mechanism of Novel Telomerase Inhibitor Nilo 22 on Leukemia Cells].

OBJECTIVE: To investigate the cytotoxic effect and its mechanism of the micromolecule compound on the leukemia cells. METHODS: The cytotoxic effects of 28 Nilotinib derivatives on K562, KA, KG, HA and 32D cell lines were detected by MTT assays, and the compound Nilo 22 was screen out. Cell apoptosis and cell cycle on leukemia cells were detected by flow cytometry. The effect of compound screened out on leukemogenesis potential of MLL-AF9 leukemia mice GFP+ cells was tested by colony-forming units assays (CFU). The cytotoxic effect was further detected by transplant assays ex vivo. Telomerase activity assay, C-circle assay were used to measure the effects of compound on the length mechanism of telomere, RT-PCR was used to detected the changes of telomere. RESULTS: Nilo 22 serves as the most outstanding candidate out of 28 Nilotinib derivatives, which impairs leukemia cell lines, but spares normal hematopoietic cell line. Comparing with Nilotinib, Nilo 22 could induce the apoptosis of GFP+ cells significantly, slightly arrests the cell cycle at G0/G1 phase, and significantly inhibits colony formation and prolong the progression in MLL-AF9 leukemia mice model. The expression showed that the compound could slow the disease progression in MLL-AF9 leukemia mice significantly. Mechanistically, Nilo 22 could reduce the length of telomere by inhibiting telomerase activity and alternative lengthening of telomere (ALT). CONCLUSION: Nilo 22 shows a significant cytotoxic effect on mice and human leukemia cells, especially for drug resistance cells. Nilo 22 is a promising anti-leukemia agent to solve the common clinical problems of drug resistance and relapse of leukemia.