hTERT Inhibition Research Articles

Favipiravir, an antiviral drug, in combination with tamoxifen exerts synergistic effect in tamoxifen-resistant breast cancer cells via hTERT inhibition

Tamoxifen (TAM) is one of the most successful treatments for breast cancer; however, TAM resistance continues to be a significant barrier. TAM resistance has been reported to be associated with increased expression of human telomerase reverse transcriptase (hTERT). This enzyme shares structural similarity with RNA-dependent RNA polymerase (RdRp) enzyme of RNA viruses, suggesting that RdRp inhibitors may also inhibit hTERT. Favipiravir (FAV) is an antiviral drug that inhibits RdRp of RNA viruses. Thus, we propose that FAV may also elicit an antitumor effect by suppressing hTERT. This study aimed to investigate the effect of FAV and TAM on TAM-resistant breast cancer (TAMR-1). The cell viabilities were determined. The levels of CDK1/ hTERT, in addition to regulators of hTERT-targeted signaling pathways were measured. Apoptosis, migration, and cell cycle distribution were also determined. Our data revealed that the combination of TAM and FAV suppressed cell proliferation synergistically (CI < 1) and resulted in a significant change in cell migration and apoptosis. Indeed, this was associated with reduced levels of hTERT and CDK1 and shift in the cell cycle distribution. Our findings suggest that the TAM/FAV combination exhibits synergistic effects against TAMR-1 human breast cancer cells by targeting hTERT.

Potential Anti-Cancer Effect of Helenalin as a Natural Bioactive Compound on the Growth and Telomerase Gene Expression in Breast Cancer Cell Line.

The telomerase gene is overexpressed in the majority of tumors and cancers compared to normal and healthy cells, and on the other hand, this enzymatic protein is overactive, therefore, the telomerase enzyme is considered a primary target for diagnostic and therapeutic purposes in most cancers. This has been hypothesized that Helenalin has anti-telomerase activity in a wide range of cancers and Tumor tissues. In this study, we investigated the inhibitory effect of helenalin extract on telomerase gene expression in the T47D breast cancer cell line. We used the MTT assay to evaluate the cytotoxic effect of different concentrations of helenalin on the T47D breast cancer cell line at 24, 48, and 72 hours. Besides, the expression of the hTERT gene in T47D cell lines treated with 1.0 and 5.0 µM helenalin after 24, 48, and 72 h incubation times was investigated through real-time PCR. According to the MTT assay, the inhibitory effect of helenalin on T47D cell proliferation is time and dose-dependent. Moreover, the results of Real-time PCR showed that exposure of T47D cell lines to helenalin led to a significant Decreasing in the expressional values of the hTERT gene as a time and dose-dependent procedure compared with the control group (P ≤ 0.05). These preliminary results demonstrated the cytotoxic potential of helenalin through inhibition of hTERT against T47D breast cancer cells.

Memantine enhances the cisplatin-induced apoptosis in A2780 ovarian cancer cells via CyclinD1 and hTERT inhibition

Cisplatin is the first-line treatment for ovarian cancer. Therefore, the use of effective compounds with synergistic effects can be considered a new strategy. Memantine is an NMDA receptors blocker which is overexpressed in cancers, including ovarian cancer and thus can be an efficient adjunct in cancer treatment. In this study, the MTT test, Flow cytometry, and DAPI staining was used to examine the combined effect of cisplatin with memantine. A study on Cyclin D1 and hTERT expression levels were conducted by qRT-PCR, and also cell cycle study of treated cells was performed. Our findings demonstrated that memantine enhances cytotoxicity in A2780 ovarian cancer cells. Apoptosis was also enhanced in cells treated with the combination of memantine and cisplatin. Memantine reduced the expression of Cyclin D1 and hTERT in A2780 cells and decreased the number of cancer cells in the cell cycle's growth and division phase. Our study suggests that memantine can be considered a potent adjuvant in the treatment of ovarian cancer.

Antithetic hTERT Regulation by Androgens in Prostate Cancer Cells: hTERT Inhibition Is Mediated by the ING1 and ING2 Tumor Suppressors.

Simple SummaryThe expression of the catalytic subunit of the human telomerase reverse transcriptase subunit (hTERT) is hormonally controlled. Androgen treatment suppresses the hTERT expression at a transcriptional level in prostate cancer cells. Here, we identified the responsive promoter element that mediates the androgen receptor induced transrepression of hTERT. The negative androgen response element (nARE) is identified as 62 bp located in the core promoter of hTERT. Chromatin immunoprecipitations indicate an androgen-dependent recruitment of the androgen receptor (AR) ING1 and ING2 to the hTERT promoter. Interestingly, the androgen-induced transrepression is mediated by the class II tumor suppressors inhibitor of growth 1 and 2, namely ING1 and ING2, respectively.The human telomerase is a key factor during tumorigenesis in prostate cancer (PCa). The androgen receptor (AR) is a key drug target controlling PCa growth and regulates hTERT expression, but is described to either inhibit or to activate. Here, we reveal that androgens repress and activate hTERT expression in a concentration-dependent manner. Physiological low androgen levels activate, while, notably, supraphysiological androgen levels (SAL), used in bipolar androgen therapy (BAT), repress hTERT expression. We confirmed the SAL-mediated gene repression of hTERT in PCa cell lines, native human PCa samples derived from patients treated ex vivo, as well as in cancer spheroids derived from androgen-dependent or castration resistant PCa (CRPC) cells. Interestingly, chromatin immuno-precipitation (ChIP) combined with functional assays revealed a positive (pARE) and a negative androgen response element (nARE). The nARE was narrowed down to 63 bp in the hTERT core promoter region. AR and tumor suppressors, inhibitor of growth 1 and 2 (ING1 and ING2, respectively), are androgen-dependently recruited. Mechanistically, knockdown indicates that ING1 and ING2 mediate AR-regulated transrepression. Thus, our data suggest an oppositional, biphasic function of AR to control the hTERT expression, while the inhibition of hTERT by androgens is mediated by the AR co-repressors ING1 and ING2.

Abstract 3108: Human telomerase reverse transcriptase regulates stemness of pancreatic cancer

Abstract Backgrounds: Human telomerase reverse transcriptase (hTERT) plays a key role on immortalization of cancer cells. In the present study, we clarified the effect of small interfering RNA (siRNA) targeting hTERT in pancreatic cancer cell lines. Methods: Two sequences of siRNA targeting hTERT and negative control siRNA were transfected into PANC-1 and MIA PaCa-2 human pancreatic cancer cell lines. Cell proliferation (WST-8 cell counting assay), stemness (sphere formation) and migration (scratch assay) were analyzed using hTERT-siRNA transfected cells and control-siRNA transfected cells. The expression levels of CDK1, SOX9 and nestin were analyzed by real time PCR. Results: As compared to control-siRNA, hTERT-siRNA suppressed cell proliferation and stemness of pancreatic cancer cell lines. Cell migration did not show any difference between hTERT-siRNA and control-siRNA transfected cells. The expression levels of CDK1, SOX9 and nestin in hTERT-siRNA transfected cells were lower than those in control-siRNA transfected cells. Conclusion: The present study indicates that inhibition of hTERT in pancreatic cancer cells suppressed stemness via alteration of the expression levels of CDK1, SOX9 and nestin. Citation Format: Juanjuan Ye, Keiko Yamakawa, Yuko Narusawa, Yoko Matsuda. Human telomerase reverse transcriptase regulates stemness of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3108.

Triptolide inhibits human telomerase reverse transcriptase by downregulating translation factors SP1 and c-Myc in Epstein-Barr virus-positive B lymphocytes.

Epstein-Barr virus (EBV) mainly causes infectious mononucleosis and is associated with several neoplasms, including Burkitt's lymphoma, nasopharyngeal carcinoma and lymphoproliferative disease. Human telomerase reverse transcriptase (hTERT) regulates enzymatic activity of telomerase and is closely associated with tumorigenesis and senescence evasion. Triptolide (TP) is a diterpenoid triepoxide, with a broad-spectrum anticancer and immunosuppressive bioactivity profile. The present study investigated whether TP inhibited hTERT expression and suppressed its activity. The mRNA and protein levels of hTERT were examined by reverse transcription-quantitative PCR and western blotting. The activity of hTERT promoter was determined by dual-luciferase reporter assay. Cell Counting Kit-8 assays were performed to analyze cell proliferation. The present study used EBV-positive B lymphoma cells as a model system, and the results demonstrated that TP significantly decreased hTERT transcription and protein expression. Mechanistically, TP attenuated the hTERT promoter activity by downregulating the expression levels of specificityprotein 1 and c-Myc transcription factors. Consistently, inhibition of hTERT via shRNA transfection efficiently enhanced the suppression of cell proliferation by TP. Furthermore, TP increased virus latent replication and promoted the lytic cycle of EBV in EBV-positive B lymphoma cells, increasing the number of lytic cells and inhibiting the viability of tumor cells. Taken together, the results of the present study revealed a molecular mechanism of the pharmacological inhibition of tumor cell proliferation by TP, encouraging the translation of TP-based therapeutics in EBV-positive B lymphoma treatment.

Exosomal miR-1255b-5p targets human telomerase reverse transcriptase in colorectal cancer cells to suppress epithelial-to-mesenchymal transition.

Cancer cells undergo epithelial-to-mesenchymal transition (EMT) in response to hypoxia. Exosomes produced in tumor microenvironments carry microRNAs (miRNAs) that affect proliferation, metastasis, and EMT. Hypoxic regulation of EMT is associated with telomerase content and stability, but the underlying mechanisms remain unclear. We identified a targeting relationship between tumor-suppressing miR-1255b-5p and human telomerase reverse transcriptase (hTERT) via clinical screening of serum samples in colorectal cancer (CRC) patients. EMT suppression via exosomal miR-1255b-5p delivery was investigated by assessing hTERT expression, Wnt/β-catenin signaling, and telomerase activity. We revealed that hypoxia directly affected exosomal miR-1255b-5p content, the delivery of which between CRC cells significantly impacted cell invasion, EMT-related protein expression, and telomerase stability. Specifically, miR-1255b-5p suppressed EMT by inhibiting Wnt/β-catenin activation via hTERT inhibition. Hypoxia reduced exosomal miR-1255b-5p secretion by CRC cells, thereby increasing hTERT expression to enhance EMT and telomerase activity. In a mouse CRC model, hypoxic exosomes containing overexpressed miR-1255b-5p attenuated EMT, tumor progression, and liver metastasis. Our results suggest the antitumor role of miR-1255b-5p and its involvement in the regulation of hTERT-mediated EMT. We propose that miRNA-targeted regulation of telomerase is a promising therapeutic strategy for future CRC treatment.

Atorvastatin-induced senescence of hepatocellular carcinoma is mediated by downregulation of hTERT through the suppression of the IL-6/STAT3 pathway

Hepatocellular carcinoma (HCC), a hepatic malignancy, has a poor prognosis and contributes to cancer-related death worldwide. Cellular senescence is an anticancer therapeutic strategy that causes irreversible cell cycle arrest and enables immune-mediated clearance of cancer cells. Atorvastatin, an HMG-CoA reductase inhibitor, has been shown to inhibit tumor growth and induce apoptosis or autophagy in malignant tumors. However, whether atorvastatin can induce HCC cell senescence and the mechanisms involved are poorly understood. The effects of atorvastatin-induced senescence were examined in both HCC cells and mouse xenograft models. The phenomenon and mechanism of senescence were examined by cell cycle analysis, senescence-associated β-galactosidase (SA-β-gal) staining and western blotting in HCC cells, and HCC tissues from mice were analyzed by immunohistochemical (IHC) staining. We demonstrated that atorvastatin induced cell growth inhibition and G0/G1 phase cell cycle arrest, leading to senescence in HCC cells. Atorvastatin-induced senescence was independent of p53, p14, and p16, and atorvastatin not only decreased the secretion of IL-6, a major senescence-associated secretory phenotype (SASP) factor, and the phosphorylation of STAT3 but also inhibited the expression of hTERT, a catalytic subunit of telomerase. Supplementation with exogenous IL-6 reversed both atorvastatin-induced suppression of STAT3 phosphorylation and hTERT expression and atorvastatin-induced senescence. Overexpression of constitutively activated STAT3 rescued HCC cells from atorvastatin-induced hTERT suppression and senescence. Moreover, atorvastatin decreased tumor growth in mouse xenograft models. Consistent with these results, atorvastatin decreased the IL-6, p-STAT3, and hTERT levels and increased β-gal expression in tumor sections. Taken together, these data indicate that atorvastatin can induce atypical cellular senescence in HCC cells to inhibit tumor growth, an effect mediated by downregulation of hTERT through suppression of the IL-6/STAT3 pathway.

Synthesis of Combretastatin A-4 and 3'-Aminocombretastatin A-4 derivatives with Aminoacid Containing Pendants and Study of Their Interaction with Tubulin and as Downregulators of the VEGF, hTERT and c-Myc Gene Expression.

Natural product combretastatin A-4 (CA-4) and its nitrogenated analogue 3′-aminocombretastatin A-4 (AmCA-4) have shown promising antitumor activities. In this study, a range of CA-4 and AmCA-4 derivatives containing amino acid pendants have been synthesized in order to compare their biological actions with those of their parent compounds. Thus, inhibition of cell proliferation on tumor cell lines HT-29, MCF-7 and A-549, as well as on the nontumor cell line HEK-273; in vitro tubulin polymerization; mitotic cell arrest; action on the microtubule cell network and inhibition of VEGF, hTERT, and c-Myc genes have been evaluated. Some AmCA-4 derivatives bearing L-amino acids exhibited inhibition of cell proliferation at low nanomolar levels exceeding the values shown by AmCA-4. Furthermore, while CA-4 and AmCA-4 derivatives do not show significant effects on the in vitro tubulin polymerization and cell cycle arrest, some selected CA-4 and AmCA-4 derivatives are able to cause total depolymerization of the microtubule network on A-549 cells. The best results were obtained in the inhibition of gene expression, particularly on the VEGF gene, in which some AmCA-4 derivatives greatly exceeded the inhibition values achieved by the parent compound.

HKR3 regulates cell cycle through the inhibition of hTERT in hepatocellular carcinoma cell lines.

Hepatocellular carcinoma is a malignant disease with improved hepatic regeneration and survival, and is activated by human telomere transferase (hTERT). hTERT is expressed during early fetal development and switched off in most adult tissues, but it becomes reactivated in HCC. The exact mechanism regulating these expression changes remains unknown during HCC progress. We evaluated the relationship between hTERT expression and human kruppel-related 3 (HKR3) and cell cycle-related factors in HCC cell lines. Following transfection for hTERT knockdown and HKR3 overexpression, proteomic and transcriptomic analyses related to hTERT were performed using liquid chromatography/mass spectrometry (LC/MS) and RNA sequencing (RNAseq) in HCC cell lines. The expression levels of hTERT, HKR3, and cell cycle-related factors were measured using western blotting, and tumor growth were evaluated via cell proliferation and cell cycle assays. Transcriptomic and proteomic analyses showed that HKR3, hTERT and cyclin-dependent kinase inhibitor 2A (CDKN2A) were correlated. Up-regulation of HKR3 expression decreased hTERT and cyclin activation and suppressed the G1/S phase of the cell cycle through CDKN2A activation. Our results suggest that HKR3 induced regulation of cell cycle through hTERT inhibition and CDKN2A activation. Our results will facilitate further exploration of the pathways regulating human telomerase activity in HCC cell lines.

Human telomerase reverse transcriptase in papillary thyroid cancer: gene expression, effects of silencing and regulation by BET inhibitors in thyroid cancer cells.

Mutations in TERT promoter have been detected in the more aggressive papillary thyroid cancers (PTCs). To elucidate the role of TERT as an eligible molecular target in these tumors, the expression of hTERT was analyzed in a series of PTCs and the effects of both pharmacological and RNA-interference-induced hTERT silencing were investigated in two human PTC cell lines (K1 and BCPAP). The expression levels of hTERT mRNA and protein were evaluated by real-time PCR and western blot assays, respectively. Effects of hTERT silencing on PTC cell lines were analyzed by MTT, migration and western blot assays. Pharmacological inhibition of hTERT was performed using two bromodomain and extra-terminal (BET) inhibitors, JQ1 and I-BET762. hTERT expression results increased in 20 out of 48 PTCs, including tumors either positive or negative for the presence of hTERT promoter and/or BRAF mutations. In K1 and BCPAP cells, hTERT silencing determined a reduction in cell viability (~50% for K1 and ~70%, for BCPAP, vs control) and migration properties that were associated with a decrease of AKT phosphorylation and β-Catenin expression. Moreover, hTERT mRNA levels were down-regulated by two BET inhibitors, JQ1 and I-BET762, which at the same dosage (0.5 and 5 µM) reduced the growth of these thyroid cancer cells. These findings demonstrate that hTERT may represent an excellent therapeutic target in subgroups of aggressive PTCs.

Nuclear localization of Desmoplakin and its involvement in telomere maintenance.

The interaction between genomic DNA and protein fundamentally determines the activity and the function of DNA elements. Capturing the protein complex and identifying the proteins associated with a specific DNA locus is difficult. Herein, we employed CRISPR, the well-known gene-targeting tool in combination with the proximity-dependent labeling tool BioID to capture a specific genome locus associated proteins and to uncover the novel functions of these proteins. By applying this research tool on telomeres, we identified DSP, out of many others, as a convincing telomere binding protein validated by both biochemical and cell-biological approaches. We also provide evidence to demonstrate that the C-terminal domain of DSP is required for its binding to telomere after translocating to the nucleus mediated by NLS sequence of DSP. In addition, we found that the telomere binding of DSP is telomere length dependent as hTERT inhibition or knockdown caused a decrease of telomere length and diminished DSP binding to the telomere. Knockdown of TRF2 also negatively influenced DSP binding to the telomere. Functionally, loss of DSP resulted in the shortened telomere DNA and induced the DNA damage response and cell apoptosis. In conclusion, our studies identified DSP as a novel potential telomere binding protein and highlighted its role in protecting against telomere DNA damage and resultant cell apoptosis.

Telomerase gene expression bioassays indicate metabolic activation of genotoxic lower chlorinated polychlorinated biphenyls

Polychlorinated biphenyls (PCBs) are ubiquitously occurring pollutants with different chemical and toxicological properties. In this study we evaluated blood plasma samples of two PCB-exposed cohorts for their ability to alter telomerase (hTERT) gene expression. Blood plasma from PCB-exposed individuals inhibited hTERT expression depending solely on the concentration of lower chlorinated PCBs, with the lowest observed adverse effect level (LOAEL) at a plasma concentration between 0.5 and 2 µg/L of LC PCBs. Individual OH-metabolites derived from the WHO indicator congeners PCB 28 and PCB 101 mimicked these effects on hTERT expression in vitro with high toxicity, including DNA damage. However, by the combination of different OH-metabolites, the bio effective PCB concentration was reduced and the respective effects on hTERT expression could be increased. At a concentration which showed no toxic activity in MTT assay, hTERT inhibition reflected the interference of OH-PCBs with the mitochondrial respiratory chain, which could lead to the production of reactive oxygen species (ROS). As individual OH-metabolites already showed a much stronger inhibition of hTERT gene expression at a lower concentration than their parental compounds, the hTERT gene expression bioassay described in this study seems to indicate metabolic activation of LC PCBs rather than the mere effect of LC PCBs on their own. In summary, this study provides dose-response linkages between effects of lower chlorinated PCBs and their concentrations in human plasma.

Abstract 4841: Prostate cancer cell death triggered by a small molecule interacting with the hTERT G-quadruplex

Abstract Human telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase, is widely overexpressed in human cancers including prostate cancer. Along with its regulation of the telomere, hTERT was also shown to regulate various signal transduction mechanisms involving DNA damage response, cell cycle checkpoint, and apoptosis. The majority of human solid tumors display telomerase activity, which was found to be well correlated with hTERT expression, and hTERT inhibition led to reduced telomerase activity and telomere length. However, approaches that directly inhibit the action of telomerase have not produced a therapeutically useful response because of long lag times for subsequent cell division to produce telomere shortening. Alternatively, recent studies demonstrated that this delay problem can be solved through downregulation of hTERT transcription. It was shown that telomeric DNA is capable of folding into four-stranded guanine quadruplex (G4) structures, which then lock the hTERT promoter activity that leads to robust telomere shortening. Our new approach has focused on the discovery of small molecules that could stabilize G-quadruplexes by interacting with the telomeric G-rich overhang stem loop, producing a lead candidate compound that enhances the kinetic folding rate of the G4 silencer element. Our in vitro studies demonstrate that this singular mechanism of action permitted reduced telomerase production and telomere shortening. Significantly, hTERT downregulation with the identified molecule caused prostate cancer cell death, which was characterized by activation of the caspase cascade and increased Annexin V- positive cells. In contrast, this compound did not harm normal prostate epithelial cells. Similarly, mouse prostate cancer cells that do not contain a G-quadruplex were not killed, demonstrating that the unique mechanism of action is mediated through the hTERT promoter G-quadruplex element. Our in vivo study demonstrated that administration of this significantly delayed tumor growth on xenografted mice. The rapid onset of cell death by G4 stands in contrast to the delayed action of classic telomerase inhibition. Therefore, our current study provides important data to support developing inhibitors of hTERT as a cancer drug target. Citation Format: Jin H. Song, Libia A. Luevano, Hyunjin Kang, Vijay Gokhale, Laurence H. Hurley, Andrew S. Kraft. Prostate cancer cell death triggered by a small molecule interacting with the hTERT G-quadruplex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4841.

Synergistic Anti-proliferative Effects of Metformin and Silibinin Combination on T47D Breast Cancer Cells via hTERT and Cyclin D1 Inhibition.

There is a growing body of data that chemotherapeutic combination strategies would be more effective in reducing drug toxicity, inhibiting tumor progression in comparison to either drug alone. To explore a chemopreventive strategy for improving breast cancer treatment efficacy, the anticancer effects of a combination of Metformin (MET) and Silibinin (SIL) were investigated in T47D breast cancer cells. Cytotoxicity of the drugs individually and in combination was evaluated using MTT assay. The precise nature of the interaction between MET and SIL was further analyzed through the median-effect method. In addition, qRT-PCR was applied to determine the expression levels of hTERT and cyclin D1 genes after 48 h drug exposure. MTT assays showed that MET and SIL individually inhibited the cell viability in a dose and time-dependent manner, and the obtained combination indices (CIs) were<1 for all the combination treatments, indicating that the anticancer agents synergistically induced growth inhibition in the breast cancer cells. qPCR findings revealed that the drug combination also synergistically down-regulated the expression levels of hTERT and cyclin D1 at all used concentrations compared with the drugs used alone after 48 h treatment (P≤0.05). The results provide evidence that synergistic antiproliferative effects of MET and SIL, linking to the down-regulation of Cyclin D1 and hTERT genes, and propose that MET+SIL may have therapeutic value in breast cancer therapy.

Telomerase reverse transcriptase interference synergistically promotes tumor necrosis factor‑related apoptosis‑inducing ligand‑induced oral squamous cell carcinoma apoptosis and suppresses proliferation invitro and invivo.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to induce cell apoptosis in many types of cancer cells. However, some malignant cells still exhibit anti-apoptosis features induced by TRAIL; thus the underlying mechanisms that regulate sensitivity and resistance of tumor cells to TRAIL-induced apoptosis remain unclear. Human telomerase reverse transcriptase (hTERT) is overexpressed in most types of human tumors and is mostly inactive in somatic cells. The present study aimed to investigate the endogenous effects and mechanisms of hTERT inhibition and TRAIL overexpression on TRAIL-induced apoptosis of human oral squamous cell carcinoma (OSCC) cells. The effects of adeno-associated virus (AAV)-mediated TRAIL and hTERT gene silencing by RNA interference were investigated on the proliferation and apoptosis of human OSCC cells in vitro and in vivo. The present results suggest that knockdown of hTERT expression accelerated TRAIL-resistant OSCC cells to TRAIL-induced apoptosis and impaired OSCC cell proliferation. In addition, this process is accompanied by the upregulation of caspase-3, caspase-8 and caspase-9, and downregulation of B cell lymphoma-2. Additionally, the possible mechanisms underlying the association between TRAIL expression and hTERT silencing were explored. The results demonstrated that TRAIL expression levels were elevated when the hTERT gene was silenced, and notable anti-tumor effects were observed when TRAIL upregulation and hTERT gene silencing were carried out simultaneously. The present findings provide experimental evidence for the combined use of TRAIL and hTERT as a possible gene therapy strategy in oral cancer.

Role of telomerase expression in T cell differentiation and proliferation

Abstract To understand the role of telomerase in T cell differentiation and function, we analyzed human telomerase reverse transcriptase (hTERT) mRNA expression and telomerase activity in six T cell subsets from 85 healthy human donors (aged 17–82 years old). We found that levels of hTERT mRNA and telomerase activity were higher in CD4 T cell subsets as compared to corresponding CD8 T cell subsets, and decreased from naïve (TN) to memory (central, TCM and effector, TEM). In all six subsets, in vitro activation with anti-CD3/CD28 antibody-coated beads led to increased amounts of hTERT mRNA (from 12 to 105-fold) as well as telomerase enzymatic activity, while the differences in hTERT amounts and telomerase activity among subsets remained in the same order observed prior to stimulation. We next analyzed the impact of different amounts of hTERT and induced telomerase activity on proliferation and lifespan of these subsets in vitro. We found that proliferation and survival were most robust in CD4 TN and poorest in CD8 TEM, correlating with their respective levels of hTERT mRNA and telomerase activity both pre-activation and over the course of a 15-day culture following a single stimulation. These findings suggest that greater hTERT expression and telomerase activity promote T cell viability and proliferative potential, and that differentiation is associated with loss of proliferative potential in T cells. We are currently analyzing whether inhibition of hTERT and telomerase activity will lead to reduced proliferation and viability in TN cells. This research was supported entirely by the Intramural Research Program of the NIH, National Institute on Aging.

Pterostilbene down-regulates hTERT at physiological concentrations in breast cancer cells: Potentially through the inhibition of cMyc.

Human telomerase reverse transcriptase (hTERT) encodes the catalytic subunit of telomerase, which has been shown to be upregulated in many cancers. Pterostilbene is a naturally occurring stilbenoid and phytoalexin found primarily in blueberries that exhibits antioxidant activity and inhibits the growth of various cancer cell types. Therefore, the aim of this study was to determine whether treatment with pterostilbene, at physiologically achievable concentrations, can inhibit the proliferation of breast cancer cells and down-regulate the expression of hTERT. We found that pterostilbene inhibits the cellular proliferation of MCF-7 and MDA-MB-231 breast cancer cells in both a time- and dose-dependent manner, without significant toxicity to the MCF10A control cells. Pterostilbene was also shown to increase apoptosis in both breast cancer cell lines. Dose-dependent cell cycle arrest in G1 and G2/M phase was observed after treatment with pterostilbene in MCF-7 and MDA-231 cells, respectively. hTERT expression was down-regulated after treatment in both a time- and dose-dependent manner. Pterostilbene also reduced telomerase levels in both cell lines in a dose-dependent manner. Moreover, cMyc, a proposed target of the pterostilbene-mediated inhibition of hTERT, was down-regulated both transcriptionally and posttranscriptionally after treatment. Collectively, these findings highlight a promising use of pterostilbene as a natural, preventive, and therapeutic agent against the development and progression of breast cancer.

The herbal medicine Melissa officinalis extract effects on gene expression of p53, Bcl-2, Her2, VEGF-A and hTERT in human lung, breast and prostate cancer cell lines

IntroductionEarlier, we verified that Melissa officinalis extract (MOE) elicits potent antiproliferative effects on different human cancer cells. To gain insights into the molecular mechanisms accounting for the cytotoxic effects of MOE, we assessed the expression patterns of several prominent molecules with therapeutic potential in cancer by Quantitative PCR (Q-PCR). MethodsA549, MCF-7 and PC3 cancer cells were grown in complete RPMI 1640 and seeded in 24 well micro plates. After incubation for 72h, 100μg/ml of MOE was added and the cells were further incubated for 72h. Afterwards, the cells were subjected to RNA extraction for the means of Q-PCR. ResultsOur results indicated that in PC3 cancer cells, MOE resulted in a significant downregulation of VEGF-A (0.0004 fold), Bcl-2 (0.001 fold), Her2 (0.02 fold), and hTERT (0.023 fold) compared to the untreated control. In addition, VEGF-A and hTERT mRNA were significantly downregulated in MCF-7 and A549 cancer cells, as well. Notably, high anti-angiogenic activity was closely associated with a high anti-telomerase activity of MOE in studying cancer cells. The decrease in VEGF-A expression was significantly superior than that of hTERT downregulation, as PC3 cancer cells with the highest hTERT down regulation (0.023) presented the highest anti VEGF activity (0.0004 fold), whereas MCF-7 cells with the lowest hTERT inhibition (0.213) showed the lowest VEGF inhibition(0.0435) among the three studied cancer cells. We noticed that the modulation of VEGF-A and hTERT gene expression can be considered as a common target, accounting for the therapeutic potential of MOE on human breast, lung and prostate cancer cells. ConclusionAltogether, it is suggested that the potent antiproliferative activity of the hydroalcoholic extract of Melissa officinalis is somehow explainable by its high potency to inhibit expression of the prominent oncogenes Bcl2, Her2, VEGF-A and hTERT in prostate cancer. In tumors with functional p53, including MCF-7 and A549 cancer cells, the role of p53, Bcl2 and Her2 is less significant. It appears that MOE exerts its antiproliferative effects in these cancer cells partly via concurrent downregulation of VEGF-A and hTERT. Additional studies are needed to clarify the role of other active molecules in cancer cells harboring functional p53.

Quercetin nanoparticles display antitumor activity via proliferation inhibition and apoptosis induction in liver cancer cells.

Quercetin is a potent cancer therapeutic agent and dietary antioxidant present in fruit and vegetables. Quercetin prevents tumor proliferation by inducing cell cycle arrest and is a well known cancer therapeutic agent and autophagy mediator. Recent studies showed that drug delivery by nanoparticles have enhanced efficacy with reduced side effects. In this regard, gold-quercetin into poly(DL-lactide-co-glycolide) nanoparticles was examined. In this study, we explored the role and possible underlying mechanisms of quercetin nanoparticle in regulation of antitumor activity in liver cancer cells. Treatment with quercetin nanoparticle effectively inhibited the liver cancer cell proliferation, cell migration and colony formation, thus suppressing liver cancer progression. Quercetin nanoparticle also upregulated apoptosis markedly. Further study suggested that quercetin nanoparticle accelerated the cleavage of caspase-9, caspase-3, and induced the up-releasing of cytochrome c (Cyto-c), contributing to apoptosis in liver cancer cells. Quercetin nanoparticles also promoted telomerase reverse transcriptase (hTERT) inhibition through reducing AP-2β expression and decreasing its binding to hTERT promoter. In addition, quercetin nanoparticle had an inhibitory role in cyclooxygenase 2 (COX-2) via suppressing the NF-κB nuclear translocation and its binding to COX-2 promoter. Quercetin nanoparticle also inactivated Akt and ERK1/2 signaling pathway. Taken together, our results suggested that quercetin nanoparticle had an antitumor effect by inactivating caspase/Cyto-c pathway, suppressing AP-2β/hTERT, inhibiting NF-κB/COX-2 and impeding Akt/ERK1/2 signaling pathways. Our results provided new mechanistic basis for further investigation of quercetin nanoparticles to find potential therapeutic strategies and possible targets for liver cancer inhibition.