hTERT Inhibition Research Articles

Inhibition of hTERT in pancreatic cancer cells by pristimerin involves suppression of epigenetic regulators of gene transcription

Inhibition of hTERT in pancreatic cancer cells by pristimerin involves suppression of epigenetic regulators of gene transcription

Induction of apoptotic endonuclease EndoG with DNA-damaging agents initiates alternative splicing of telomerase catalytic subunit hTERT and inhibition of telomerase activity hTERT in human CD4+ and CD8+ T-lymphocytes

Activity of telomerase catalytic subunit hTERT (human Telomerase Reverse Transcriptase) can be regulated by alternative splicing of its mRNA. At present time exact mechanism of hTERT splicing is not fully understood. Apoptotic endonuclease EndoG is known to participate this process. EndoG expression is induced by DNA damages. The aim of this work was to investigate the ability of DNA-damaging agents with different mechanism of action to induce EndoG expression and inhibit telomerase activity due to the activation of hTERT alternative splicing in normal activated human CD4+ and CD8+ T-lymphocytes. All investigated DNA-damaging agents were able to induce EndoG expression. Cisplatin, a therapeutic compound, producing DNA cross-links induced the highest level of DNA damages and EndoG expression. Incubation of CD4+ and CD8+ T-cells with cisplatin caused the changes in proportion of hTERT splice variants and inhibition of telomerase activity.

A Pharmacological Chaperone Molecule Induces Cancer Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters.

Activation of human telomerase reverse transcriptase (hTERT) is necessary for limitless replication in tumorigenesis. Whereas hTERT is transcriptionally silenced in normal cells, most tumor cells reactivate hTERT expression by alleviating transcriptional repression through diverse genetic and epigenetic mechanisms. Transcription-activating hTERT promoter mutations have been found to occur at high frequencies in multiple cancer types. These mutations have been shown to form new transcription factor binding sites that drive hTERT expression, but this model cannot fully account for differences in wild-type (WT) and mutant promoter activation and has not yet enabled a selective therapeutic strategy. Here, we demonstrate a novel mechanism by which promoter mutations activate hTERT transcription, which also sheds light on a unique therapeutic opportunity. Promoter mutations occur in a core promoter region that forms tertiary structures consisting of a pair of G-quadruplexes involved in transcriptional silencing. We show that promoter mutations exert a detrimental effect on the folding of one of these G-quadruplexes, resulting in a nonfunctional silencer element that alleviates transcriptional repression. We have also identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, GTC365, that acts at an early step in the G-quadruplex folding pathway to redirect mutant promoter G-quadruplex misfolding, partially reinstate the correct folding pathway, and reduce hTERT activity through transcriptional repression. This transcription-mediated repression produces cancer cell death through multiple routes including both induction of apoptosis through inhibition of hTERT's role in regulating apoptosis-related proteins and induction of senescence by decreasing telomerase activity and telomere length. We demonstrate the selective therapeutic potential of this strategy in melanoma cells that overexpress hTERT.

Fucoidan Induces ROS-Dependent Apoptosis in 5637 Human Bladder Cancer Cells by Downregulating Telomerase Activity via Inactivation of the PI3K/Akt Signaling Pathway.

Preclinical Research Fucoidan, a sulfated polysaccharide, is a compound found in various species of seaweed that has anti-viral, anti-bacterial, anti-oxidant, anti-inflammatory, and immunomodulatory activities; however, the underlying relationship between apoptosis and anti-telomerase activity has not been investigated. Here, we report that fucoidan-induced apoptosis in 5637 human bladder cancer cells was associated with an increase in the Bax/Bcl-2 ratio, the dissipation of the mitochondrial membrane potential (MMP, Δψm), and cytosolic release of cytochrome c from the mitochondria. Under the same experimental conditions, fucoidan-treatment decreased hTERT (human telomerase reverse transcriptase) expression and the transcription factors, c-myc and Sp1. This was accompanied by decreased telomerase activity. Fucoidan-treatment also suppressed activation of the PI3K/Akt signaling pathway. Inhibition of PI3K/Akt signaling enhanced fucoidan-induced apoptosis and anti-telomerase activity. Meanwhile, fucoidan treatment increased the generation of intracellular ROS, whereas the over-elimination of ROS by N-acetylcysteine, an anti-oxidant, attenuated fucoidan-induced apoptosis, inhibition of hTERT, c-myc, and Sp1 expression, and reversed fucoidan-induced inactivation of the PI3K/Akt signaling pathway. Collectively, these data indicate that the induction of apoptosis and the inhibition of telomerase activity by fucoidan are mediated via ROS-dependent inactivation of the PI3K/Akt pathway. Drug Dev Res 78 : 37-48, 2017. © 2016 Wiley Periodicals, Inc.

Telomere-Mitochondrion Links Contribute to Induction of Senescence in MCF-7 Cells after Carbon-Ion Irradiation.

The effects of carbon-ion irradiation on cancer cell telomere function have not been comprehensively studied. In our previous report cancer cells with telomere dysfunction were more sensitive to carbon-ion irradiation, but the underlying mechanisms remained unclear. Here we found that telomerase activity was suppressed by carbon-ion irradiation via hTERT down-regulation. Inhibition of telomere activity by MST-312 further increased cancer cell radiosensitivity to carbon-ion radiation. hTERT suppression caused by either carbon-ion irradiation or MST-312 impaired mitochondrial function, as indicated by decreased membrane potential, mtDNA copy number, mitochondrial mass, total ATP levels and elevated reactive oxygen species (ROS). PGC-1α expression was repressed after carbion-ion irradiation, and hTERT inhibition by MST-312 could further exacerbate this effect. Lowering the mitochondrial ROS level by MitoTEMPO could partially counteract the induction of cellular senescence induced by carbon-ion radiation and MST-312 incubation. Taken together, the current data suggest that telomere-mitochondrion links play a role in the induction of senescence in MCF-7 cells after carbon-ion irradiation.

Nrf2-driven TERT regulates pentose phosphate pathway in glioblastoma.

Given the involvement of telomerase activation and dysregulated metabolism in glioma progression, the connection between these two critical players was investigated. Pharmacological inhibition of human Telomerase reverse transcriptase (hTERT) by Costunolide induced glioma cell apoptosis in a reactive oxygen species (ROS)-dependent manner. Costunolide induced an ROS-dependent increase in p53 abrogated telomerase activity. Costunolide decreased Nrf2 level; and ectopic Nrf2 expression decreased Costunolide-induced ROS generation. While TERT knock-down abrogated Nrf2 levels, overexpression of Nrf2 increased TERT expression. Inhibition of hTERT either by Costunolide, or by siRNA or dominant-negative hTERT (DN-hTERT) abrogated (i) expression of Glucose-6-phosphate dehydrogenase (G6PD) and Transketolase (TKT) – two major nodes in the pentose phosphate (PPP) pathway; and (ii) phosphorylation of glycogen synthase (GS). hTERT knock-down decreased TKT activity and increased glycogen accumulation. Interestingly, siRNA-mediated knock-down of TKT elevated glycogen accumulation. Coherent with the in vitro findings, Costunolide reduced tumor burden in heterotypic xenograft glioma mouse model. Costunolide-treated tumors exhibited diminished TKT activity, heightened glycogen accumulation, and increased senescence. Importantly, glioblastoma multiforme (GBM) patient tumors bearing TERT promoter mutations (C228T and C250T) known to be associated with increased telomerase activity; exhibited elevated Nrf2 and TKT expression and decreased glycogen accumulation. Taken together, our findings highlight the previously unknown (i) role of telomerase in the regulation of PPP and glycogen accumulation and (ii) the involvement of Nrf2-TERT loop in maintaining oxidative defense responses in glioma cells.

Inhibition of human telomerase reverse transcriptase in vivo and in vitro for retroviral vector-based antisense oligonucleotide therapy in ovarian cancer.

Human telomerase is absent in most normal tissues, but is abnormally activated in all major cancer cells. Telomerase enables tumor cells to maintain telomere length, allowing indefinite replicative capacity. Albeit not sufficient in itself to induce neoplasia, telomerase is believed to be necessary for cancer cells to grow without limit. Studies using an antisense oligonucleotide (ASODN) to the RNA component of telomerase or human telomerase reverse transcriptase (hTERT) demonstrate that telomerase in human tumor lines can be blocked in vivo. Inhibition of hTERT led to telomere shortening and cancer cell death, validating telomerase as a target for anticancer genetic therapy. Varieties of approaches for hTERT inhibition have been investigated. The aim of this study was to analyze the biological activity of ASODN to the hTERT mediated by retrovirus vector, which was used as therapy for ovarian tumor. We constructed and characterized a recombinant retrovirus vector with full-length hTERT antisense complementary DNA. The vector was introduced into ES-2 by lipofectamine-mediated gene transfection. The cellular proliferation and telomerase activity of the transformant cells were retarded. The hTERT gene expression and the telomerase activity of the transformant cells were both decreased. The transformant cells show partial reversion of the malignant phenotype. PT67 cells were also transfected with the recombinant vector and virus-producer cells were generated. The retrovirus-containing supernatant effectively inhibited the growth of human ovarian tumor xenografts in mouse models (subcutaneous tumor model), and enhanced the mouse survival time.

Bortezomib-mediated down-regulation of telomerase and disruption of telomere homeostasis contributes to apoptosis of malignant cells.

Bortezomib inhibits the ubiquitin/proteasome pathway to achieve its anti-cancer effect and its well characterized activity is the NF-κB inhibition through which the anti-apoptotic bcl-2 expression is down-regulated and apoptosis is subsequently induced. However, the downstream molecular targets of bortezomib are still incompletely defined. Because telomere stabilization via activation of telomerase, induction of telomerase reverse transcriptase (hTERT) and appropriate expression of shelterin proteins is essential to cancer development and progression, we investigated the effect of bortezomib on telomere homeostasis/function in malignant cells. The bortezomib treatment of leukemic (HEL) and gastric cancer cells (BGC-823) led to significant inhibition of hTERT and telomerase expression, widespread dysregulation of shelterin protein expression, and telomere shortening, thereby triggering telomere dysfunction and DNA damage. hTERT over-expression attenuated bortezomib-induced telomere shortening, abnormal shelterin expression and telomere dysfunction. Importantly, bortezomib-mediated apoptosis of malignant cells was partially prevented by hTERT over-expression. Mechanistically, hTERT first robustly enhances bcl2 expression and maintains significantly high residual levels of bcl2 even in bortezomib-treated HEL cells. Second, hTERT protects against bortezomib-induced DNA damage. Our findings collectively reveal a profound impact of bortezomib on telomere homeostasis/function. Down-regulation of hTERT expression and telomere dysfunction induced by bortezomib both contribute to its cancer cell killing actions. It is evident from the present study that hTERT can confer resistance of malignant cells to bortezomib-based target cancer therapy, which may have important clinical implications.

327 - Histone Carbonylation Is a Novel Redox-Regulated Epigenomic Modification in Adipose Tissue of Obese C57/Bl6 Mice

The effects of polyunsaturated fatty acids (PUFAs) obtained from the diet on colorectal cancer have been widely explored. However, controversial results have been obtained about the role played by the lipid peroxidation products of PUFAs, such as 4-hydroxy-nonenal (HNE), in the control of colon cancer growth. This aldehyde, indeed, showed both procarcinogenic and protective effects. In an attempt to verify the action of HNE, we studied the effects of a low dose of HNE (1 μM), similar to those “physiologically” found in normal cells and plasma, on telomerase activity, a key parameter of malignant transformation. Caco-2 cells were exposed to HNE and, paralleling cell growth inhibition, we observed the down-regulation of telomerase activity and hTERT expression. Similar effects have also been observed in HT-29 cells, in which HNE inhibited cell proliferation, telomerase activity and hTERT expression, suggesting that the inhibition of telomerase activity could be a general mechanism involved in the antiproliferative effect exerted by this aldehyde. Finally, we elucidated the mechanism of hTERT inhibition by HNE. A reduction of GSH content preceded the decrease of telomerase activity, but this only partially explained the telomerase activity inhibition. The major mechanism of HNE action seems to be the modulation of expression and activity of transcription factors belonging to the Myc/Mad/Max network.Since the presence of PUFAs in the diet exposes epithelial colon cells to HNE, this aldehyde could contribute to cell growth control through the inhibitory action on telomerase activity and hTERT expression, suggesting a protective effect on colon mucosa.

Abstract 3799: Cordyceptin induces apoptosis through repressing hTERT expression and inducing extranuclear export of hTERT

Abstract Cordycepin is an adenosine analog originally extracted from Cordyceps militaris that possesses many pharmacological effects including immune activation and antioxidant and antitumor effects. However, the underlying relationship between apoptosis and telomerase activity in response to cordycepin exposure has not been investigated. In this study, we found that cordycepin-induced apoptosis of human leukemia cells (H937 and THP-1 cells) was associated with inactivation of telomerase and downregulation of human telomerase reverse transcriptase (hTERT) as well as the transcription factors c-Myc and Sp1, which are required for basal transcription from the hTERT gene promoter. Cordycepin also attenuated the activation of phosphoinositide-3-kinase (PI3K)/Akt signaling, thereby reducing phosphorylation and nuclear translocation of hTERT. We further showed that the PI3K inhibitor LY29004 significantly decreased telomerase activity in cordycepin-treated cells and increased cordycepin-induced cell death. These findings demonstrate that cordycepin is cytotoxic to human leukemia cells and suppresses telomerase activity through transcriptional and post-translational suppression of hTERT by inactivating the PI3K/Akt signaling pathway. Citation Format: Nam Deuk Kim, Gi-Young Kim, Yung Hyun Choi. Cordyceptin induces apoptosis through repressing hTERT expression and inducing extranuclear export of hTERT. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3799. doi:10.1158/1538-7445.AM2015-3799

Knockdown of hTERT and Treatment with BIBR1532 Inhibit Cell Proliferation and Invasion in Endometrial Cancer Cells.

Telomerase activity and expression of the catalytic protein hTERT are associated with cell proliferation and advanced stage in endometrial cancer. Our objective was to evaluate the effect of inhibition of hTERT by siRNA and BIBR1532 on cell growth, apoptosis and invasion in endometrial cancer cells. Knockdown of hTERT or treatment of the cells with BIBR1532 decreased telomerase activity, inhibited cell proliferation, induced apoptosis, and reduced cell invasion in Ishikawa and ECC-1 cells. Either hTERT siRNA or BIBR1532 in combination with paclitaxel promoted a synergistic inhibitory effect on cell growth through induction of Annexin V expression and a remarkable reduction in cell invasion through reduction of protein expression of MMP9, MMP2, and MMP3. Increased telomerase activity and hTERT protein expression by transfections enhanced the protein expression of MMPs and increased the cell invasion ability. BIBR1532 significantly antagonized cell invasion induced by increased hTERT expression. These findings suggest that telomerase and hTERT facilitate cell invasion via MMP family in human endometrial cancer cells.

Inhibition of hTERT and telomerase activity via hampering MAPK kinase signaling in renal cell carcinoma cell lines.

e13501 Background: Telomerase adds species specific telomeric repeats onto the ends of chromosomes. Inhibition of telomerase and Human telomerase reverse transcriptase (hTERT) is an exciting therapeutic target since it is required for the long term proliferation of most cancer. In this study we use hTERT inhibition as a anticancer approach in renal cell carcinoma. Methods: 82 cases of RCC were included in the present study. Relative telomerase activity was measured by Telo TAGGG Telomerase PCR ELISA PLUSsupplied by Roche applied science as per the manufacturer’s instruction. Gene expression of hTERT was estimated by real time PCR. RCC cell lines ACHN and A498 were used in the study. Activator and inhibitor of MAPK pathway EGF and U0126 was used at 80ng/ml and 20µM concentration respectively. To confirm the activation and inhibition of MAPK pathway phosphorylation of ERK was checked by RayBio cell based ERK1/2 (Thr202/Tyr204) ELISA kit as per manfacturer’s instructions. Effect of hTERT inhibition on cancer properties of RCC cell lines ACHN & A498 was tested by MTT assay, Propidium iodide staining and cytofluorimetric analysis. Results: The expression of hTERT and telomerase activity were significantly higher in the tumor tissue in comparison with normal renal parenchyma (p=0.000). Cell proliferation was inhibited significantly after treatment with U0126 in ACHN and A498 cells as compare to untreated cells (p=0.001). Cell cycle analysis by Propidium iodide staining showed that the growth of the U0126 cells was arrested in SubG0/G1 phase (p=0.001).Whereas Cytofluorimetric analysis of the AnnexinV-FITC versus PI staining revealed that U0126 treatment induced apoptosis in ACHN and A498 cells (p=0.001). Conclusions: Our result suggests the expression of hTERT was significantly higher in tumor tissue as compare to normal renal tissue. Inhibition of hTERT, drove RCC cancer cells into crisis and death. Thus inhibition of hTERT via inhibition of MAPK pathway may offer new opportunities for realizing the promise of telomerase inhibition therapy.

HTERT Inhibition Triggers Epstein–Barr Virus Lytic Cycle and Apoptosis in Immortalized and Transformed B Cells: A Basis for New Therapies

Induction of viral lytic cycle, which induces death of host cells, may constitute a useful adjunct to current therapeutic regimens for Epstein-Barr virus (EBV)-driven malignancies. Human telomerase reverse transcriptase (hTERT), essential for the oncogenic process, may modulate the switch from latent to lytic infection. The possible therapeutic role of hTERT inhibition combined with antiviral drugs was investigated. EBV-negative BL41 and convertant EBV-positive BL41/B95.8 Burkitt's lymphoma cell lines and lymphoblastoid cell lines (LCL) were infected with retroviral vector encoding short hairpin RNA (shRNA) anti-hTERT and cultured with or without the prodrug ganciclovir. The effects on EBV lytic replication, cell proliferation, and apoptosis were characterized. hTERT silencing by shRNA induced the expression of BZLF1, EA-D, and gp350 EBV lytic proteins and triggered a complete lytic cycle. This effect was associated with downregulation of BATF, a negative regulator of BZLF1 transcription. hTERT silencing also resulted in antiproliferative and proapoptotic effects. In particular, hTERT inhibition induced an accumulation of cells in the S-phase, an effect likely due to the dephosphorylation of 4E-BP1, an AKT1-dependent substrate, which results in a decreased availability of proteins needed for cell-cycle progression. Besides inducing cell death through activation of complete EBV lytic replication, hTERT inhibition triggered AKT1/FOXO3/NOXA-dependent apoptosis in EBV-positive and -negative Burkitt's lymphoma cells. Finally, ganciclovir enhanced the apoptotic effect induced by hTERT inhibition in EBV-positive Burkitt's lymphomas and LCLs. These results suggest that combination of antiviral drugs with strategies able to inhibit hTERT expression may result in therapeutically relevant effects in patients with EBV-related malignancies.

Abstract 582: hTERT inhibition stimulates cyclooxygenase2 (COX2) expression in cancer cells and synergizes with celecoxib to exert anti-cancer effects.

Abstract Telomerase and telomerase reverse transcriptase (hTERT) confer cancer cells sustained proliferation and survival potentials through telomere lengthening dependent and independent activities. Targeting telomerase or hTERT is thus a novel anti-cancer strategy. However, telomerase/hTERT inhibition alone has minimal clinical efficacy. We explored the relationship between hTERT and oncogenic cyclooxygenase 2 (COX2) and evaluated the synergistic anti-cancer effects of targeting both hTERT and COX2. hTERT depletion in gastric and cervical cancer cells led to robust increases in mRNA and protein levels of COX2. The COX2 promoter activity increased substantially in hTERT-depleted cancer cells. Mechanistically, hTERT depletion led to the activation of p38 MAPK that was responsible for the stimulation of COX2 gene transcription. hTERT depletion or the COX2 inhibitor celecoxib alone did not affect gastric cancer cell survival, while their combination synergistically killed them both in vitro and in vivo. Taken together, hTERT induces COX2 expression in cancer cells and simultaneously targeting hTERT and COX2 synergistically kills cancer cells, which may help path a new avenue in telomerase-based cancer therapeutics. Citation Format: Tiantian Liu, Magnus Björkholm, Dawei Xu. hTERT inhibition stimulates cyclooxygenase2 (COX2) expression in cancer cells and synergizes with celecoxib to exert anti-cancer effects. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 582. doi:10.1158/1538-7445.AM2013-582 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Apigenin decreases cell viability and telomerase activity in human leukemia cell lines

Recent studies have shown that apigenin (4′,5,7-trihydroxyflavone inhibits human malignant cancer cell growth through cell cycle arrest and apoptosis. However, the underlying relationship between apoptosis and telomerase activity in response to apigenin exposure is not well understood. In this study, we found that apigenin significantly induces direct cytotoxicity in human leukemia cells (U937, THP-1 and HL60) through activation of the caspase pathway. As we presumed, treatment with apigenin was found to increase the level of intracellular reactive oxygen species (ROS), whereas pretreatment with antioxidants, N-acetyl-cysteine (NAC) or glutathione (GSH), completely attenuated ROS generation. Surprisingly, these antioxidants did not promote recuperation from apigenin-induced cell death. We further showed that apigenin downregulates telomerase activity in caspase-dependent apoptosis and observed that apigenin dosing results in downregulation of telomerase activity by suppression of c-Myc-mediated telomerase reverse transcriptase (hTERT) expression. In addition, treatment of apigenin-dosed cells with the two antioxidants did not restore telomerase activity. Taken together, this data suggests that ROS is not essential for suppression of apigenin-mediated apoptosis associated with the activation of caspases and regulation of telomerase activity via suppression of hTERT. We conclude that apigenin has a direct cytotoxic effect and the loss of telomerase activity in leukemia cells.

Abstract 3079: Loss of β2-spectrin may lead to the activation of telomerase reverse transcriptase (TERT) and hepatocellular cancer formation

Abstract Background and Aims: Telomerase is a specialized reverse transcriptase (TERT) that synthesizes repeat DNA sequences at telomeres- the specialized ends of chromosomes is suppressed in normal cells by multiple proteins that include TGF-β and Smad3 resulting in the progressive shortening of telomeres with each cell division, ultimately leading to chromosomal instability and cellular replicative senescence or growth arrest. Emerging evidence indicates that β2-spectrin (β2SP), a Smad3/Smad4 adaptor protein required for TGF-β signaling, is a powerful tumor suppressor. β2SP+/−, β2SP+/−/Smad3+/− and β2SP+/−/Smad4+/− mice dramatically develop Hepatic cancers. Because TERT is markedly activated in β2SP+/−/Smad3+/− hepatocellular cancer tissues compared to Smad3−/− tissues, we hypothesized that β2SP interaction could be involved in the interaction with Smad3 to suppress TERT expression in normal tissues, and the loss of β2SP may lead to telomerase gene activation resulting in HCC. Results: We tested human TERT (hTERT) expression levels in several HCC cell lines that have different levels of β2SP. hTERT expression levels negatively correlate with β2SP by Western Blot analysis. Loss of β2SP in β2SP+/− and β2SP−/− mouse embryonic fibroblasts (MEFs) significantly activated mTERT expression. Co-transfection of a β2SP expression plasmid and hTERT promoter-luciferase construct significantly inhibited the hTERT promoter in β2SP-deficient SNU-398 cells. Depletion of Smad3 by in vitro RNAi and the expression of hTERT-luciferase in Smad3 depleted cells upon co-transfection with Smad3 and/or β2SP expression plasmids suggest that both Smad3 and β2SP are required for the suppression of hTERT. Furthermore, chromatin immunoprecipitation (ChIP) assay suggests binding of Smad3/β2SP protein to hTERT promoter. Conclusions: Taken together our data suggest that divergent pathways converge on β2SP and Smad3 for the regulation of TERT. Activation of the TGF-β signaling pathway with suppression of TERT provides a strong strategy for generating targeted therapeutics to these lethal human cancers. 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 3079.

RNA interference-mediated hTERT inhibition enhances TRAIL-induced apoptosis in resistant hepatocellular carcinoma cells

TRAIL has been reported to induce apoptosis in a variety of tumor cell types including hepato-cellular carcinoma (HCC) cell lines. However, considerable numbers of HCC cells, especially some highly malignant tumors, show resistance to TRAIL-induced apoptosis. The molecular mechanisms that regulate sensitivity versus resistance of tumor cells to TRAIL-induced apoptosis remain poorly defined. It has been shown that human telomerase catalytic subunit (hTERT) is overexpressed in human HCCs. In this study, we investigated the effects and the mechanisms of hTERT RNAi on the TRAIL-induced apoptosis of HCC cells that exhibit resistance to TRAIL. Our results indicate that hTERT RNAi sensitizes TRAIL-resistant HCC cells to TRAIL-induced apoptosis. hTERT RNAi-mediated sensitization to TRAIL-induced apoptosis is accompanied up-regulation of procaspases-8 and -9, inhibition of telomerase activity and loss of telomere length. Our results suggest that hTERT RNAi overcame the resistance of the HCC cells against TRAIL, at least in part, via the mitochondrial type II apoptosis pathway and telomerase-dependent pathway.

Inhibition of telomerase activity alters tight junction protein expression and induces transendothelial migration of HIV-1-infected cells

Telomerase, via its catalytic component telomerase reverse transcriptase (TERT), extends telomeres of eukaryotic chromosomes. The importance of this reaction is related to the fact that telomere shortening is a rate-limiting mechanism for human life span that induces cell senescence and contributes to the development of age-related pathologies. The aim of the present study was to evaluate whether the modulation of telomerase activity can influence human immunodeficiency virus type 1 (HIV-1)-mediated dysfunction of human brain endothelial cells (hCMEC/D3 cells) and transendothelial migration of HIV-1-infected cells. Telomerase activity was modulated in hCMEC/D3 cells via small interfering RNA-targeting human TERT (hTERT) or by using a specific pharmacological inhibitor of telomerase, TAG-6. The inhibition of hTERT resulted in the upregulation of HIV-1-induced overexpression of intercellular adhesion molecule-1 via the nuclear factor-kappaB-regulated mechanism and induced the transendothelial migration of HIV-1-infected monocytic U937 cells. In addition, the blocking of hTERT activity potentiated a HIV-induced downregulation of the expression of tight junction proteins. These results were confirmed in TERT-deficient mice injected with HIV-1-specific protein Tat into the cerebral vasculature. Further studies revealed that the upregulation of matrix metalloproteinase-9 is the underlying mechanisms of disruption of tight junction proteins in hCMEC/D3 cells with inhibited TERT and exposed to HIV-1. These results indicate that the senescence of brain endothelial cells may predispose to the HIV-induced upregulation of inflammatory mediators and the disruption of the barrier function at the level of the brain endothelium.

Growth Arrest in Ovarian Cancer Cells by hTERT Inhibition Short-Hairpin RNA Targeting Human Telomerase Reverse Transcriptase Induces Immediate Growth Inhibition but not Necessarily Induces Apoptosis in Ovarian Cancer Cells

ABSTRACTIt is believed that telomerase is a terminal effector and its role is to maintain the length of telomeres. However, the main rate-limiting subunit of telomerase, human telomerase reverse transcriptase (hTERT), has been recently proved to have the ability to affect a variety of other genes such as p53 independently of the decurtation of telomere. To test whether this extra-telomeric effect could contribute to additional and immediate alterations in cellular proliferation and apoptosis, three epithelial ovarian cancer cell lines (A2780 [wild-type p53], OVCAR3 [mutant p53], and SKOV3 [p53 null]) were treated with hTERT-targeted short-hairpin RNA (shRNA), and their short-term effects were evaluated. A very fast growth inhibiting response to downregulation of hTERT was observed in all three cell lines with S-phase arrest karyotypes. But apoptosis appeared only in A2780 and OVCAR3 cells with p53 accumulation and elevated p21. In contrast, a decrease in expression of p21 was observed in SKOV3 and cell death appeared to be unaffected. These results indicated for the first time that inhibition of hTERT could induce immediate growth arrest in human ovarian carcinoma cells by blocking them in S phase, but apoptosis may only be induced via extra-telomeric effects on activation of p53 and p21.

Simultaneous inhibition of both hTERT and androgen receptor gene expression in LNCaP cells using single shRNA vector

To explore the feasibility of inhibition of hTERT and androgen receptor (AR) gene expression simultaneously in LNCaP cells by single shRNA vector. Templates DNA of both hTERT and AR siRNA were inserted into Pgenesil vector to construct a new vector Pgenesil-hTERT-AR-shRNA by RNAi-DNA vector technology. Pgenesil-HK-shRNA, Pgenesil-hTERT-shRNA, Pgenesil-AR-shRNA and Pgenesil-hTERT-AR-shRNA vectors were transfected into prostate cancer LNCaP cells respectively. The levels of AR mRNA, apoptosis and proliferation of each cell group were determined by FQ-PCR, Annexin V method and MTT. The level of hTERT mRNA of control group cells and cells transfected by Pgenesil-HK-shRNA, Pgenesil-hTERT-shRNA, Pgenesil-AR-shRNA and Pgenesil-hTERT-AR-shRNA was (1.51 +/- 0.08) x 10(8), (7.32 +/- 0.43) x 10(7), (2.94 +/- 0.15) x 10(6), (4.45 +/- 0.25) x 10(7) and (3.17 +/- 0.18) x 10(6) (copies/ml) respectively. The level of AR mRNA of control cell groups and cells transfected by Pgenesil-HK-shRNA, Pgenesil-hTERT-shRNA, Pgenesil-AR-shRNA and Pgenesil-hTERT-AR-shRNA was (1.92 +/- 0.11) x 10(5), (6.47 +/- 0.32) x 10(5), (3.70 +/- 0.24) x 10(4), (1.22 +/- 0.06) x 10(4) and (7.21 +/- 0.41) x 10(3) (copies/ml) respectively. These data indicate that the expression of hTERT or AR gene could be significantly inhibited by Pgenesil-hTERT-shRNA or Pgenesil-AR-shRNA while Pgenesil-hTERT-AR-shRNA could simultaneously inhibit both hTERT and AR gene expression. The apoptosis rate and the inhibition rate of cell growth of Pgenesil-hTERT-AR-shRNA group were significantly higher than those of Pgenesil-hTERT-shRNA group or Pgenesil-AR-shRNA group (P < 0.05). It is feasible to inhibit both hTERT and AR gene expression simultaneously by single shRNA vector. It will be a new research strategy of gene therapy for prostate cancer.