Inhibition Of hTERT Expression Research Articles

Abstract 656: A novel dual-targeting peptide HS1002 enhances anti-tumor activity via GnRH/human telomerase reverse transcriptase (hTERT) interaction in human prostate cancer cells

Abstract Human telomerase reverse transcriptase (hTERT) is overexpressed in most human cancers. hTERT is considered as an important target for cancer therapy. In the present study, a new peptide HS1002 were synthesized with sequence of pGlu-His-Trp-Ser-Tyr-Arg-Leu-Arg-Phe-Ile-Pro-NHEt based on the GnRH agonist and hTERT. The aim of this study was to evaluate HS1002 for its anticancer activity by dual-targeting to GnRH/hTERT in prostate cancer cells. Upon comparison with different prostate cancer cell lines, HS1002 exhibited highest cytotoxicity against LNCaP cells. The hTERT expression correlated with telomerase activity in LNCaP cells, was significantly inhibited by HS1002, resulting in reduction of proliferation, metastasis and increase in apoptosis. Additionally, HS1002 suppressed c-Myc and AKT/ERK pathway in LNCaP cells. In the tumor-bearing nude mice model, HS1002 significantly inhibited tumor growth and downregulated the expression of hTERT and Ki-67 in tumor tissue as evidenced by western blot and immunohistochemistry analysis, respectively. Moreover, repeated subcutaneous injection of HS1002 resulted in the reduction of both serum testosterone levels and seminal vesicle weight. HS1002 also increased cytosolic calcium influx and CRE-luciferase activity in GnRHR-overexpressing HEK293 cells compared to mock-transfected transfected cells. Furthermore, a potent cytotoxicity in LNCaP cells was exhibited by HS1002-activated peripheral blood mononuclear cell (PBMC) with interleukin (IL)-2 using LDH release assay. In addition, a flow-cytometric analysis confirmed an elevation in the production of granzyme B and IFN-γ in CD8+ T cells in the MC38 syngeneic mouse model by HS1002. These data indicate that the inhibition of hTERT expression by HS1002 suppresses prostate cancer cell growth and induced anti-cancer immunity. Therefore, HS1002 might be used as a novel therapeutic drug for prostate cancer. Citation Format: Jae Hyeon Park, Ju Ri Kim, Hwa Young Cha, Hyung Sik Kim. A novel dual-targeting peptide HS1002 enhances anti-tumor activity via GnRH/human telomerase reverse transcriptase (hTERT) interaction in human prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 656.

Anti-Cancer Role of Dendrosomal Nano Solanine in Chronic Myelogenous Leukemia Cell Line through Attenuation of PI3K/AKT/mTOR Signaling Pathway and Inhibition of hTERT Expression.

Solanine was primarily known as a toxic compound. Nonetheless, recently the apoptotic role of solanine through suppression of PI3K/AKT/mTOR signaling pathway has been shown against many malignancies except chronic myelogenous leukemia (CML). Sustaining the aforementioned pro-survival pathway, BCR-ABL fused oncoprotein in CML activates NF-kB and c- MYC for apparent immortalizing factor hTERT. Since solanine is a poor water-soluble molecule, herein, a nanocarrier was employed to intensify its pernicious effect on cancerous cells. The current research aimed at evaluating the effect of dendrosomal nano solanine (DNS) on leukemic and HUVEC cells. DNS characterization was determined by NMR, DLS and TEM. The viability, apoptosis and cell cycle of DNS and imatinib-treated cells were determined. A quantitative real-time PCR was employed to measure the expression of PI3K, AKT, mTOR, S6K, NF-kB, c-MYC and hTERT mRNAs. The Protein levels were evaluated by western blot. Investigating the anticancer property of free and dendrosomal nano solanine (DNS) and the feasible interplaying between DNS and imatinib on leukemic cells, we figured out the potential inhibitory role of DNS and DNS+IM on cancerous cells in comparison with chemotherapy drugs. Moreover, results revealed that the encapsulated form of solanine was much more preventive on the expression of PI3KCA, mTOR, NF-kB, c-MYC and hTERT accompanied by the dephosphorelating AKT protein. The results advocate the hypothesis that DNS, rather than solanine, probably due to impressive penetration, can restrain the principal pro-survival signaling pathway in erythroleukemia K562 and the HL60 cell lines and subsequently declined mRNA level of hTERT which causes drug resistance during long-term treatment. Additionally, combinational treatment of DNS and IM could also bestow an additive anti-leukemic effect. As further clinical studies are necessary to validate DNS efficacy on CML patients, DNS could have the potency to be considered as a new therapeutic agent even in combination with IM.

Interaction studies of carbon nanomaterials and plasma activated carbon nanomaterials solution with telomere binding protein

Most cancer cells have telomerase activity because they can express the human telomerase reverse transcriptase (hTERT) gene. Therefore, the inhibition of the hTERT expression can play an important role in controlling cancer cell proliferation. Our current study aims to inhibit hTERT expression. For this, we synthesized graphene oxide (GO) and a functionalized multiwall carbon nanotube (f-MWCNT), latter treated them with cold atmospheric pressure plasma for further analysis of the hTERT expression. The inhibition of hTERT expression by GO, f-MWCNT, plasma activated GO solution (PGOS), and plasma activated f-MWCNT solution (PCNTS), was studied using two lung cancer cell lines, A549 and H460. The hTERT experimental results revealed that GO and PGOS sufficiently decreased the hTERT concentration, while f-MWCNT and PCNTS were unable to inhibit the hTERT concentration. Therefore, to understand the inhibition mechanism of hTERT, we studied the binding properties of GO and PGOS with telomere binding protein (AtTRB2). The interaction studies were carried out using circular dichroism, fluorescence, 1H-15N NMR spectroscopy, and size-exclusion chromatography (SEC) binding assay. We also used docking simulation to have an better understanding of the interactions between GO nanosheets and AtTRB2 protein. Our results may provide new insights that can benefit in biomedical treatments.

Inhibition of hTERT expression by MAP kinase inhibitor induces cell death in renal cell carcinoma

BackgroundHuman telomerase reverse transcriptase (hTERT) is one of the components of telomerase enzyme and its activity is associated with cell proliferation and differentiation. Extracellular signal regulated kinase (ERK)-mitogen activated protein kinase signaling pathway play an important role in hTERT expression. The present study was conducted to ascertain hTERT messenger RNA (mRNA) expression in renal cell carcinoma (RCC) and its association with clinicopathological characteristics. Further, we also explored hTERT targeting as possible tumor therapeutic. MethodsA total of 96 histopathologically confirmed RCC cases and corresponding normal tissues were subjected to hTERT gene expression using real-time PCR. Two RCC cell lines viz. ACHN and A498 were treated with MEK inhibitor (U0126) and hTERT mRNA expression, telomerase activity, cell viability, migration, and apoptosis were evaluated. ResultsThe hTERT mRNA levels were found to be significantly higher in RCC as compared with corresponding normal renal tissues (P = 0.040) as well as in high grades to that of low grades clear cell RCC (P = 0.008). Significantly diminished ERK phosphorylation and hTERT mRNA expression concomitantly reduced telomerase activity that was observed after U0126 treatment. Subsequently, cell viability and migration were significantly inhibited after treatment with U0126 in both the cell lines to that of control (P = 0.001). In addition, U0126-treated cells showed significantly increased apoptosis (P = 0.001) to that of controls. ConclusionHenceforth, this study infer that, hTERT expression can be used as a possible diagnostic marker for RCC, and inhibition of hTERT expression by hampering ERK-mitogen activated protein kinase cascade may be used as a promising anticancer target in RCC.

Abstract PR02: Specific inhibition of hTERT expression in melanoma by targeting common promoter mutations which cause quadruplex DNA instability

Abstract Background: Telomerase reverse transcriptase (hTERT) is a catalytic subunit of the enzyme telomerase. It has recently been shown that the hTERT promoter is commonly mutated (>75% of cases) in malignant melanoma. These mutations occur at four sites in a G-rich region of the promoter which has previously been shown to form quadruplex DNA and to downregulate hTERT expression. We have tested the hypothesis that mutations in the quadruplex-forming region of the hTERT promoter destabilize quadruplex-formation resulting in increased hTERT expression and cellular proliferation, thus providing a novel therapeutic target for melanoma. Materials and Methods: Quadruplex formation by the mutated and wild type hTERT promoter oligonucleotides was determined by circular dichroism. Analytical ultracentrifugation was used for sedimentation equilibrium analysis. Thermal denaturation was used to characterize the relative stability of the mutated and wild type oligonucleotides. The growth inhibitory effect of mutated and wild type oligonucleotides were determined in four cell lines which contained mutated or wild type hTERT promoter sequences. Cell proliferation was characterized by MTT and cell counting. Results: In order to characterize the effects of the hTERT mutations, the biophysical properties of structures formed by wild-type and mutant TERT sequences were explored by several methods. Circular dichroism and thermal denaturation studies showed that all sequences formed quadruplex structures but that those formed by the mutated sequences were markedly less stable than the wild-type. Analytical ultracentrifugation showed that all sequences formed one major unimolecular folded form but that mutant sequences had a greater tendency to form misfolded aggregated species than the wild-type. Addition of the quadruplex binder TmPyP4 to the mutant sequences lessened that amount of such aggregates and resulted in sedimentation profiles that closely resembled the wild-type sequences. Treatment of cells with mutated hTERT promoter sequence with oligonucleotides encoding the mutated or wild type sequence resulted in significant growth inhibition that was time and concentration dependent. DNA crosslinking studies indicate that the G-rich oligonucleotide is binding to the C-rich strand by Watson-Crick base pairing suggesting that it inhibits transcription initiation by strand invasion. A similar growth inhibitory effect was seen in cells exposed to quadruplex stabilizing agents. Conclusions: The common mutations in the hTERT promoter destabilize quadruplex formation and likely prevent quadruplex-mediated transcriptional silencing. This instability can be overcome by quadruplex-binding drugs and/or oligonucleotides encoding this sequence. The growth of cell lines containing the hTERT promoter mutations is inhibited by oligonucleotides encoding the wild type and mutated sequences. These oligonucleotides stabilize the mutated genomic quadruplex structure, resulting in transcriptional silencing. This approach has obvious clinical potential. This abstract is also being presented as Poster A07. Citation Format: Donald M. Miller, Alexandra Sokolova, Francine Rezzoug, Shelia Thomas, Jonathan Chaires, Jonathan Chaires, William Dean, John Trent. Specific inhibition of hTERT expression in melanoma by targeting common promoter mutations which cause quadruplex DNA instability. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr PR02.

372 Specific inhibition of hTERT expression by targeting common promoter mutations which cause quadruplex DNA instability

372 Specific inhibition of hTERT expression by targeting common promoter mutations which cause quadruplex DNA instability

Abstract 3206: A novel prodrug of the green tea polyphenol (−)-epigallocatechin-3-gallate inhibits the telomerase gene by modifying DNA methylation and histone acetylation in breast cancer cells

Abstract Epigallocatechin-3-gallate (EGCG), a major component of green tea polyphenols (GTPs), has been reported to down-regulate telomerase activity in breast cancer cells thereby increasing cellular apoptosis and inhibiting cellular proliferation. However, the major concerns with GTPs and EGCG are the bioavailability and poor stability under physiological conditions. In the present study, we have observed that treatments with EGCG and a novel pro-drug of EGCG (Pro-EGCG) dose-dependently inhibit the cellular proliferation of human breast carcinoma MCF-7 and MDA-MB-231 cells. Inhibition of telomerase has received wide attention in breast cancer prevention because of its unique expression in cancer cells and low level of expression in normal mammary epithelial cells. Both EGCG and Pro-EGCG inhibited the transcription of hTERT (human telomerase reverse transcriptase) by epigenetic modification in MCF-7 and MDA-MB-231 cells. The inhibition of hTERT expression was consistently correlated with hTERT promoter hypomethylation and deacetylation. Chromatin immunoprecipitation (ChIP) analysis of the hTERT promoter revealed that EGCG and Pro-EGCG increased the level of inactive trimethyl H3K9, but decreased active dimethyl H3K4 and acetyl H3K9. In addition, EGCG and Pro-EGCG significantly inhibited DNA methyltransferase (DNMT) activity, which might have an important role in the down-regulation of hTERT by demethylation of the CpG sites in the hTERT promoter in breast cancer cells. Bisulfite sequencing of the hTERT promoter confirmed the induced hypomethylation of the hTERT promoter by EGCG and Pro-EGCG. Down-regulation of hTERT was also mediated through significant inhibition of histone acetyltransferase (HAT), an enzyme responsible for histone acetylation, by EGCG and Pro-EGCG. Histone deacetylase (HDAC) activity was not altered by EGCG and Pro-EGCG, which reveals that EGCG- and Pro-EGCG-induced histone deacetylation was mediated through inhibition of HAT, but not by effects on HDACs in these breast cancer cells. These results show that EGCG and Pro-EGCG are inhibiting the cellular proliferation and inducing apoptosis in these breast carcinoma cells but not in normal control mammary epithelial MCF10A cells, at least in part, through epigenetic modification of telomerase expression. Further, our results also show that Pro-EGCG is more stable than EGCG and required a lesser dose than EGCG to facilitate the same cellular and epigenetic effects in breast carcinoma cells. Collectively, our data provide new insights into breast cancer prevention through epigenetic modulation of telomerase by using Pro-EGCG, a more stable form of EGCG, as a novel chemopreventive drug. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3206.

Abstract 3204: The mRNA decay factor tristetraprolin (TTP) induces senescence in human papillomavirus-transformed cervical cancer cells by targeting E6-AP ubiquitin ligase

Abstract Cervical cancer is the second most common cancer among women worldwide. A necessary factor in the development of nearly all cases of cervical cancer is the human papillomavirus (HPV) infection. High-risk HPV (HPV16 and 18) mediates cellular transformation and carcinogenesis through the concerted actions of viral oncoproteins E6 and E7 that disrupt the normal cell cycle regulation by targeting the p53 and Rb tumor suppressor pathways respectively. In the present study, we found that expression of the mRNA decay factor TTP is lost in HPV18-positive HeLa cells and introduction of TTP in cells lead to inhibition of cell proliferation and tumorigenesis by inducing cellular senescence. Consistent with this, TTP-expressing cells showed enhanced p53 protein stability and p53-dependent transcriptional activity. In cells expressing TTP, no changes were observed in the RNA levels of E6 and E7 indicating that these viral factors are not targets of TTP-mediated RNA decay. Whereas the E6-associated protein (E6-AP), the cellular ubiquitin ligase required for E6-mediated p53 degradation, was found to be down regulated both at mRNA and protein levels in presence of TTP. Furthermore, the association of E6 and E6-AP promotes transcriptional activation of hTERT, and TTP-dependent down regulation of E6-AP resulted in inhibition of hTERT expression and decreased cellular telomerase activity. Analysis of E6-AP mRNA revealed potential AU-rich element (ARE) binding sites of TTP present in the 3’ untranslated region (3′UTR) and RNA-binding studies demonstrate a physical interaction between TTP and E6-AP mRNA leading to rapid decay of E6-AP mRNA in a 3’UTR-dependent manner. Similar results were obtained with other high-risk HPV-positive cell lines that employ E6-AP pathway to control p53 and hTERT levels. Consistent with this, we found TTP expression to be lost or significantly reduced in squamous cell carcinomas as compared to normal cervical tissue. Finally, our studies indicate that loss of TTP expression in cervical cancer occurs through epigenetic mechanisms involving promoter methylation and histone deacetylation. These findings demonstrate the ability of TTP to act as a tumor suppressor through inhibition of the E6-AP pathway and indicate TTP loss to be a critical event during HPV-mediated carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3204.

Alpha anomer of 5-aza-2′-deoxycytidine down-regulates hTERT mRNA expression in human leukemia HL-60 cells

DNA methylation inhibitors are being extensively studied as potential anticancer agents. In the present study, we compared the capability of alpha anomer of 5-aza-2′-deoxycytidine (α-5-azadCyd) to induce down-regulation of hTERT expression in HL-60 cells with other nucleoside analogs that act as DNA methylation inhibitors: β-5-azadCyd (decitabine), (S)-9-(2,3-dihydroxypropyl)adenine [(S)-DHPA], isobutyl ester of (R,S)-3-(adenin-9-yl)-2-hydroxypropanoic acid [(R,S)-AHPA-ibu] and prospective DNA methylation inhibitors (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine [(S)-HPMPazaC] and 5-fluoro-zebularine (F-PymRf). Exposure to α-5-azadCyd induced the down-regulation of hTERT expression in low micromolar concentrations (0.05–50μM). A more cytotoxic beta anomer caused a transient up-regulation of hTERT and a subsequent reduction in hTERT mRNA levels at concentrations more than 10 times below its GIC50 value. In this respect, (S)-DHPA and (R,S)-AHPA-ibu were less efficient, since a similar effect was achieved at concentrations above their GIC50. In contrast, F-PymRf treatment resulted in up to a three-fold induction of hTERT expression within a broad range of concentrations. In all cases, the down-regulation of hTERT expression was concentration-dependent. The correlation was found between c-myc overexpression and transiently elevated hTERT expression after treatment with all tested compounds except for α-5-azadCyd and (S)-HPMPazaC. Although the primary task of hypomethylating agents in anticancer therapy lies in reactivation of silenced tumour-suppressor genes, the inhibition of hTERT expression might also be a fruitful clinical effect of this approach.

Intensive inhibition of hTERT expression by a ribozyme induces rapid apoptosis of cancer cells through a telomere length-independent pathway

Restoration of telomerase activity is essential for most of the malignancies. Telomerase reverse transcriptase (TERT) is the key component of telomerase. In this study, we designed a hammerhead ribozyme against human telomerase reverse transcriptase (hTERT) and observed its growth inhibition and pro-apoptosis effects on cancer cells. The efficiency of this ribozyme was verified in in vitro cleavage experiment. A recombinant retrovirus was constructed to transduce the ribozyme to telomerase positive colon carcinoma cell line SW480 and gastric carcinoma cell line SGC7901. We found that the ribozyme could strongly inhibit hTERT expression and telomerase activity, resulting in rapid apoptosis of cancer cells. Shortening of telomere and replicative senescence were not observed before cell death, indicating intensive inhibition of hTERT expression can induce apoptosis by some mechanism(s) except telomere shortening and replicative senescence. This study suggests that hTERT exerts a direct antiapoptotic function in cancer cells. Anti-hTERT ribozyme might be a potential means in the therapy of telomerase-positive malignancies.

Upstream stimulatory factor (USF) as a transcriptional suppressor of human telomerase reverse transcriptase (hTERT) in oral cancer cells

Telomerase activity is suppressed in normal human somatic tissues but is activated in cancer cells and immortal cell lines. The reverse transcriptase (RT) subunit human telomerase reverse transcriptase (hTERT) is the key regulator of telomerase activity. The hTERT promoter contains E-box elements and may allow upstream stimulatory factor (USF), a basic helix-loop-helix (bHLH) leucine zipper family proteins, to bind and regulate the expression. In this study, we investigated whether and how USF effect on hTERT. Through luciferase reporter assays, we found that both USF1 and USF2 possess a comparable effect on the inhibition of hTERT expression. Immunoprecipitation (IP) and immunoblotting (IB) analysis reveal that the suppression of hTERT by USF was not through the interaction of USF with c-myc or mad, nor disturbed the cellular protein levels of those. In gel mobility shift and chromatin immunoprecipitation (CHIP) assays, we found that the USF suppression is through direct binding at the E-box site of hTERT promoter and rendering the effect actively. Analysis on clinical normal and tumor tissues reveal that the expression of USF1 and USF2 was lower in the tumor tissues, correlated with hTERT expression and telomerase activity. Taking together, our results demonstrate that USF is a negative transcriptional repressor for hTERT in oral cancer cells. It is possible that USF lose the inhibitory effect on hTERT expression leading to telomerase reactivation and oral carcinogenesis.