Down-regulation Of Telomerase Research Articles

Cytotoxic and Anti-proliferative Effects of Moringa oleifera Lam. on HeLa Cells

Background:The aim of the study was to determine the mechanism of Moringa oleifera-induced apoptosis in HeLa cells. HeLa cells over-express cyclin E and cyclin B1, abrogate G0-G1 and G2-M cell cycle arrest, promoting tumorigenesis. Cyclin E, cyclin B1, E2F1 and telomerase expression, and caspase-3 and -7 activation were assessed after 24-treatment with M. oleifera leaf fractions.Material and methods:Apoptosis through caspase-3 and caspase-7 activation was determined quantitatively by the FAM FLICA™ Caspase-3/7 assay. Cyclin E, cyclin B1 and E2F1 were quantified by flow cytometry. Telomerase was evaluated by Telomeric repeat amplification protocol (TRAP reaction). The effects on colony formation were assessed by seeding treated cells in six-well plates for 7 days under culture conditions. The MTT assay was used to determine cell survival.Results:HeLa cells treated for 24 hours with M. oleifera leaf fractions showed dose-dependent cytotoxicity, activation of caspases-3 and -7, down regulation of cyclin E, cyclin B1, E2F1, and inhibition of telomerase expression. Cell cycle analysis of the dead cell population showed G2-M cellcycle arrest.Conclusion:M. oleifera leaf fractions triggered apoptosis through the mitochondrial pathway and cell cycle arrest at G2-M phase in HeLa cells after 24-hour treatment, through down regulation of cyclin E and cyclin B1 expression, and caspase-3 and -7 activation. In addition, M. oleifera leaf extract induces senescence in HeLa cells through the down-regulation of telomerase. Colony formation and cell proliferation were inhibited in a dose-dependent manner, corresponding with telomerase inhibition.

Antitumor efficacy of a recombinant EGFR-targeted fusion protein conjugate that induces telomere shortening and telomerase downregulation

ObjectiveTo prepare a recombinant EGFR-targeted fusion protein drug conjugate acting on telomere and telomerase; and evaluate its antitumor efficacy. MethodsWe prepared a recombinant fusion protein Fv-LDP-D3 which consists of the Fv fragment of an anti-EGFR monoclonal antibody (MAb), the apoprotein of lidamycin (LDP), and the third domain (D3) of human serum albumin (HSA); then generated the conjugate Fv-LDP-D3∼AE by integrating the active enediyne chomophore (AE) of lidamycin. Accordingly, in vitro and in vivo experiments were performed. ResultsAs shown, Fv-LDP-D3 specifically bound to EGFR highly-expressing cancer cells and intensely entered K-Ras mutant cells via enhanced macropinocytosis. By in vivo imaging, Fv-LDP-D3 displayed intense accumulation and persistent retention in tumor-site. Furthermore, the conjugate Fv-LDP-D3∼AE displayed highly potent cytotoxicity to cancer cells with IC50 at 0.1 nM level. The conjugate induced telomere shortening and downregulation of telomerase and EGFR pathway related proteins. Fv-LDP-D3∼AE exhibited prominent antitumor efficacy against human colorectal cancer xenograft accompanying with significant increase of serum IFN-β in athymic mice. ConclusionThe recombinant fusion protein conjugate that exhibits the capability of tumor-targeting drug delivery can induce telomere shortening and telomerase downregulation. The investigation may lay the foundation for the development of MAb-HSA domain-based fusion protein drug conjugates.

To Fix or Not to Fix: Maintenance of Chromosome Ends Versus Repair of DNA Double-Strand Breaks.

Early work by Muller and McClintock discovered that the physical ends of linear chromosomes, named telomeres, possess an inherent ability to escape unwarranted fusions. Since then, extensive research has shown that this special feature relies on specialized proteins and structural properties that confer identity to the chromosome ends, thus allowing cells to distinguish them from intrachromosomal DNA double-strand breaks. Due to the inability of conventional DNA replication to fully replicate the chromosome ends and the downregulation of telomerase in most somatic human tissues, telomeres shorten as cells divide and lose this protective capacity. Telomere attrition causes the activation of the DNA damage checkpoint that leads to a cell-cycle arrest and the entering of cells into a nondividing state, called replicative senescence, that acts as a barrier against tumorigenesis. However, downregulation of the checkpoint overcomes this barrier and leads to further genomic instability that, if coupled with re-stabilization of telomeres, can drive tumorigenesis. This review focuses on the key experiments that have been performed in the model organism Saccharomyces cerevisiae to uncover the mechanisms that protect the chromosome ends from eliciting a DNA damage response, the conservation of these pathways in mammals, as well as the consequences of their loss in human cancer.

Knockdown of the stem cell marker Musashi-1 inhibits endometrial cancer growth and sensitizes cells to radiation

BackgroundEndometrial carcinoma is the most common gynecological cancer in Europe. Musashi-1 is known to be a key regulator of endometrial cancer stem cells and a negative prognostic marker. In the present study, we aimed to understand growth and gene expression patterns in endometrial carcinoma after Musashi-1 knockdown in vitro and in vivo. Changes in therapeutic resistance were also assessed.MethodsFirst, we performed analyses to understand Musashi-1 expression patterns using The Cancer Genome Atlas database. We then proceeded to assess effects of small interfering RNA-based Musashi-1 targeting in two endometrial carcinoma cell lines, Ishikawa and KLE. After quantifying baseline changes in cell metabolism, we used MTT tests to assess chemotherapy effects and colony formation assays to understand changes in radioresistance. For mechanistic study, we used quantitative polymerase chain reaction (qPCR) and western blotting of key Musashi-1 target genes and compared results to primary tissue database studies. Finally, xenograft experiments in a mouse model helped understand in vivo effects of Musashi-1 knockdown.ResultsMusashi-1 is aberrantly expressed in primary tumor tissues. In vitro, silencing of Musashi-1 resulted in a strong decline in cell proliferation and radioresistance, while chemoresistance remained unchanged. Loss of Musashi-1 led to downregulation of telomerase, DNA-dependent protein kinase, the Notch pathway and overexpression of cyclin-dependent kinase inhibitor p21, the latter of which we identified as a key mediator of Msi-1 knockdown-related anti-proliferative signaling. In vivo, the anti-proliferative effect was confirmed, with Msi-1 knockdown tumors being about 40% reduced in size.ConclusionsMusashi-1 knockdown resulted in a strong decrease in endometrial cancer proliferation and a loss of radioresistance, suggesting therapeutic potential.

Cycloastragenol and Astragaloside IV activate telomerase and protect nucleus pulposus cells against high glucose-induced senescence and apoptosis.

In diabetes-induced intervertebral disc degeneration (Db-IVDD), senescence and apoptosis of nucleus pulposus cells (NPCs) are major contributing factors. Telomere attrition and telomerase downregulation are some of the main reasons for senescence and eventual apoptosis. The derivatives of the Chinese herb Astragalus membranaceus, Cycloastragenol (CAG) and Astragaloside IV (AG-IV), are reportedly effective telomerase activators against telomere shortening; however, their effect in Db-IVDD have not been explored. The present study simultaneously investigated the regulation of these derivatives on senescence, apoptosis, telomeres and telomerase a model of high-glucose (HG)-induced stress using rat primary NPCs. The NPCs were stimulated with HG (50 mM) to evoke HG-induced stress, and the effects of CAG and AG-IV were observed on: i) The expression level of senescence marker p16; ii) β-Gal staining; iii) the expression levels of apoptosis markers cleaved-caspase 3 (c-C3), BAX and Bcl-2; iv) telomerase activation with telomerase reverse transcriptase (TERT) mRNA and protein expression, while telomere length was measured with reverse transcription-quantitative PCR. Cell proliferation was determined using the Cell Counting Kit-8 assay. Results demonstrated an upregulation in the expression levels of p16, c-C3 and BAX, and increased β-Gal staining; while the expression level of Bcl-2 was downregulated in a concentration-dependent manner. Pre-treatment of the NPCs with CAG and AG-IV downregulated the protein expression levels of p16, c-C3 and BAX, and decreased the percentage of β-Gal and FITC staining; while upregulating the Bcl-2 expression. These effects protected the cells from HG stress-induced senescence and apoptosis. HG also downregulated the expression profile of TERT and shortened the telomere length in a glucose concentration-dependent manner. While pretreatment with CAG and AG-IV upregulated TERT expression and ameliorated the telomere attrition. CAG and AG-IV also increased cell proliferation and improved cell morphology in HG conditions. Overall, these findings indicated that CAG and AG-IV suppressed HG stress-induced senescence and apoptosis, in addition to enhancing telomerase activation and lengthening of the Telomere. Therefore, CAG and AG-IV prolonged the replicative capability and longevity of the NPCs and they have the potential to be therapeutic agents in Db-IVDD.

Epigenetic features in regulation of telomeres and telomerase in stem cells.

The epigenetic nature of telomeres is still controversial and different human cell lines might show diverse histone marks at telomeres. Epigenetic modifications regulate telomere length and telomerase activity that influence telomere structure and maintenance. Telomerase is responsible for telomere elongation and maintenance and is minimally composed of the catalytic protein component, telomerase reverse transcriptase (TERT) and template forming RNA component, telomerase RNA (TERC). TERT promoter mutations may underpin some telomerase activation but regulation of the gene is not completely understood due to the complex interplay of epigenetic, transcriptional, and posttranscriptional modifications. Pluripotent stem cells (PSCs) can maintain an indefinite, immortal, proliferation potential through their endogenous telomerase activity, maintenance of telomere length, and a bypass of replicative senescence in vitro. Differentiation of PSCs results in silencing of the TERT gene and an overall reversion to a mortal, somatic cell phenotype. The precise mechanisms for this controlled transcriptional silencing are complex. Promoter methylation has been suggested to be associated with epigenetic control of telomerase regulation which presents an important prospect for understanding cancer and stem cell biology. Control of down-regulation of telomerase during differentiation of PSCs provides a convenient model for the study of its endogenous regulation. Telomerase reactivation has the potential to reverse tissue degeneration, drive repair, and form a component of future tissue engineering strategies. Taken together it becomes clear that PSCs provide a unique system to understand telomerase regulation fully and drive this knowledge forward into aging and therapeutic application.

Long-lived termite kings and queens activate telomerase in somatic organs.

Kings and queens of termites, like queens of other advanced eusocial insects, are endowed with admirable longevity, which dramatically exceeds the life expectancies of their non-reproducing nest-mates and related solitary insects. In the quest to find the mechanisms underlying the longevity of termite reproductives, we focused on somatic maintenance mediated by telomerase. This ribonucleoprotein is well established for pro-longevity functions in vertebrates, thanks primarily to its ability of telomere extension. However, its participation in lifespan regulation of insects, including the eusocial taxa, remains understudied. Here, we report a conspicuous increase of telomerase abundance and catalytic activity in the somatic organs of primary and secondary reproductives of the termite Prorhinotermes simplex and confirm a similar pattern in two other termite species. These observations stand in contrast with the telomerase downregulation characteristic for most adult somatic tissues in vertebrates and also in solitary insects and non-reproducing castes of termites. At the same time, we did not observe caste-specific differences in telomere lengths that might explain the differential longevity of termite castes. We conclude that although the telomerase activation in termite reproductives is in line with the broadly assumed association between telomerase and longevity, its direct phenotypic impact remains to be elucidated.

HTERT Downregulation Attenuates Resistance to DOX, Impairs FAK-Mediated Adhesion, and Leads to Autophagy Induction in Breast Cancer Cells.

Telomerase is known to contribute to telomere maintenance and to provide cancer cell immortality. However, numerous reports are showing that the function of the enzyme goes far beyond chromosome ends. The study aimed to explore how telomerase downregulation in MCF7 and MDA-MB-231 breast cancer cells affects their ability to survive. Consequently, sensitivity to drug resistance, proliferation, and adhesion were assessed. The lentiviral-mediated human telomerase reverse transcriptase (hTERT) downregulation efficiency was performed at gene expression and protein level using qPCR and Western blot, respectively. Telomerase activity was evaluated using the Telomeric Repeat Amplification Protocol (TRAP) assay. The study revealed that hTERT downregulation led to an increased sensitivity of breast cancer cells to doxorubicin which was demonstrated in MTT and clonogenic assays. During a long-term doubling time assessment, a decreased population doubling level was observed. Interestingly, it did not dramatically affect cell cycle distribution. hTERT downregulation was accompanied by an alteration in β1-integrin- and by focal adhesion kinase (FAK)-driven pathways together with the reduction of target proteins phosphorylation, i.e., paxillin and c-Src. Additionally, autophagy activation was observed in MDA-MB-231 cells manifested by alternations in Atg5, Beclin 1, LC3II/I ratio, and p62. These results provide new evidence supporting the possible therapeutic potential of telomerase downregulation leading to induction of autophagy and cancer cells elimination.

Telomerase reverse transcriptase downregulation by RNA interference modulates endoplasmic reticulum stress and mitochondrial energy production

Telomerase is a cancer promoting ribonucleoprotein complex and is a potential therapeutic target for cancer. In this study, the effects of telomerase downregulation on the whole cell proteome were investigated. Understanding how the effect of downregulation on the whole proteome profile will generate a greater understanding of the possible roles played by telomerase in cancer.Downregulation was achieved by RNA interference (RNAi), targeting the telomerase reverse transcriptase (TERT) subunits of telomerase. Transfection of TERT siRNA downregulates TERT gene expression and induced downregulation of telomerase activity. Investigation of the effect of silencing TERT in telomerase was further validated through proteomic analysis by performing 2-dimension electrophoresis (2DE) coupled with MALDI-TOF/TOF. 12 protein spots in HeLa cells were reported to be significantly differentially expressed with 11 of them were upregulated and 1 downregulated. Through STRING analysis, differentially expressed proteins demonstrated strong associations with endoplasmic reticulum stress marker and mitochondrial energy production marker.In conclusions, the result exhibited novel integrated proteomic response involving endoplasmic reticulum stress and mitochondrial energy production in response to the TERT downregulation in cervical cancer cells.

"Is the Widening of Pulse Pressure and Hypertension in the Elderly Due in Large Measure to an Unrecognized Hypomagnesemia, Downregulation of Telomerase and Progressive Release of Ceramides and Platelet-Activating Factor?"

It is generally agreed that age-related increases in arterial blood pressure (ABP) are mainly a reflection of an increase in systolic..

Loss of atrx cooperates with p53-deficiency to promote the development of sarcomas and other malignancies.

The SWI/SNF-family chromatin remodeling protein ATRX is a tumor suppressor in sarcomas, gliomas and other malignancies. Its loss of function facilitates the alternative lengthening of telomeres (ALT) pathway in tumor cells, while it also affects Polycomb repressive complex 2 (PRC2) silencing of its target genes. To further define the role of inactivating ATRX mutations in carcinogenesis, we knocked out atrx in our previously reported p53/nf1-deficient zebrafish line that develops malignant peripheral nerve sheath tumors and gliomas. Complete inactivation of atrx using CRISPR/Cas9 was lethal in developing fish and resulted in an alpha-thalassemia-like phenotype including reduced alpha-globin expression. In p53/nf1-deficient zebrafish neither peripheral nerve sheath tumors nor gliomas showed accelerated onset in atrx+/- fish, but these fish developed various tumors that were not observed in their atrx+/+ siblings, including epithelioid sarcoma, angiosarcoma, undifferentiated pleomorphic sarcoma and rare types of carcinoma. These cancer types are included in the AACR Genie database of human tumors associated with mutant ATRX, indicating that our zebrafish model reliably mimics a role for ATRX-loss in the early pathogenesis of these human cancer types. RNA-seq of p53/nf1- and p53/nf1/atrx-deficient tumors revealed that down-regulation of telomerase accompanied ALT-mediated lengthening of the telomeres in atrx-mutant samples. Moreover, inactivating mutations in atrx disturbed PRC2-target gene silencing, indicating a connection between ATRX loss and PRC2 dysfunction in cancer development.

Telomere shortening in breast cancer cells (MCF7) under treatment with low doses of the benzylisoquinoline alkaloid chelidonine.

Telomeres, the specialized dynamic structures at chromosome ends, regularly shrink with every replication. Thus, they function as an internal molecular clock counting down the number of cell divisions. However, most cancer cells escape this limitation by activating telomerase, which can maintain telomere length. Previous studies showed that the benzylisoquinoline alkaloid chelidonine stimulates multiple modes of cell death and strongly down-regulates telomerase. It is still unknown if down-regulation of telomerase by chelidonine boosts substantial telomere shortening. The breast cancer cell line MCF7 was sequentially treated with very low concentrations of chelidonine over several cell passages. Telomere length and telomerase activity were measured by a monochrome multiplex quantitative PCR and a q-TRAP assay, respectively. Changes in population size and doubling time correlated well with telomerase inhibition and telomere shortening. MCF7 cell growth was arrested completely after three sequential treatments with 0.1 μM chelidonine, each ending after 48 h, while telomere length was reduced to almost 10% of the untreated control. However, treatment with 0.01 μM chelidonine did not have any apparent consequence. In addition to dose and time dependent telomerase inhibition, chelidonine changed the splicing pattern of hTERT towards non-enzyme coding isoforms of the transcript. In conclusion, telomere length and telomere stability are strongly affected by chelidonine in addition to microtubule formation.

Correction to: MYC-dependent downregulation of telomerase by FLT3 inhibitors is required for their therapeutic efficacy on acute myeloid leukemia.

The original version of this article contained a mistake. The name of Magnus Björkhom should have been Magnus Björkholm.

MYC-dependent downregulation of telomerase by FLT3 inhibitors is required for their therapeutic efficacy on acute myeloid leukemia

The somatic mutation of FLT3 occurs in 30% of acute myeloid leukemia (AML), with the majority of mutations exhibiting internal tandem duplication (ITD). On the other hand, the induction of telomerase reverse transcriptase (hTERT) and the activation of telomerase is a key step in AML development. Here, we sought to determine whether FLT3ITD regulates hTERT expression in AML cells and whether hTERT expression affects FLT3 inhibitors’ therapeutic efficacy on AML. FLT3ITD-harboring AML cell lines and primary cells treated with the FLT3 inhibitor PKC412 displayed a rapid decline in the levels of hTERT mRNA and telomerase activity. Moreover, PKC412 inhibited hTERT gene transcription in a c-MYC-dependent manner. The ectopic expression of hTERT significantly attenuated the apoptotic effect of PKC412 on AML cells. Mechanistically, hTERT enhanced the activity of FLT3 downstream effectors or alternative RTK signaling, thereby enhancing AKT phosphorylation, in AML cells treated with PKC412. Collectively, PKC412 downregulates hTERT expression and telomerase activity in a MYC-dependent manner and this effect is required for its optimal anti-AML efficacy, while hTERT over-expression confers AML cells resistance to a targeted therapeutic agent PKC412. These findings suggest that the functional interplay between FLT3ITD and hTERT contributes to the AML pathogenesis and interferes with the efficacy of FLT3ITD-targeted therapy.

Novel pan PI3K inhibitor-induced apoptosis in APL cells correlates with suppression of telomerase: An emerging mechanism of action of BKM120

The intertwining between cancer pathogenesis and perturbation of multitude signaling pathways ushered the cancer therapeutic approaches into an unbounded route of targeted therapies. For the nonce and among the plethora of promising inhibitors, intense interest has focused on small molecules targeting different component of PI3K axis. Intrigued by the constant activation of PI3K in leukemia, this study aimed to investigate the effects of BKM120, as the excelled member of pan PI3K inhibitors, in a panel of hematologic malignant cell lines. The resulting data showed that BKM120 exerted a concentration-dependent growth suppressive effect; however, IC50 values varied among the tested cells. Our results outlined that the blockage of PI3K in NB4, as the most sensitive cell line, resulted in a caspase-3-dependent apoptosis probably through NFκB-mediated suppression of c-Myc and hTERT. As far we are aware, to date, there have been no reports of BKM120 effect on enzymatic repression of telomerase, and this study represents for the first time that the anti-proliferative effect of the inhibitor on NB4 is mediated by down-regulation of telomerase; shedding new light on the novel mechanism of action of BKM120.

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.

Telomerase modulation in therapeutic approach.

Telomerase is a specialized enzymatic complex responsible for the synthesis of telomeric repeats 5'-TTAGGG-3' localized at the ends of eukaryotic chromosomes. This mechanism prevents shortening of telomeres after each cell division. The enzyme is detected in about 85% of human tumors, but it is not expressed in normal cells or its expression is significantly lower. Consequently, it provides the cancer cells immortality. Thus, since showing cancer cell specificity (to a certain extent), the enzyme became a target for an adjuvant cancer therapy. So far, in vitro studies and preclinical studies seem to be promising. This work focuses on the pathways and mechanisms that are targeted in order to eliminate telomerase with consequence of cancer cell death. The anti-telomerase strategy may be beneficial especially in the context of sensitization of tumor cell to chemotherapeutic agents. We also indicate potential side effects and consequences of telomerase downregulation that should be considered when anti-telomerase strategy is undertaken. Alternatively, we also emphasize potential useful application of telomerase induction.

Induction of Apoptosis in Pancreatic Cancer Cells by CDDO-Me Involves Repression of Telomerase through Epigenetic Pathways.

Reactivation of telomerase in cancers provides an attractive target for developing novel agents to selectively destroy tumor cells. Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me), a synthetic oleanane triterpenoid, inhibited cell proliferation and induced apoptosis in pancreatic cancer cells at very low concentrations. The antiproliferative and apoptosis-inducing effects of CDDO-Me were associated with the inhibition of human telomerase reverse transcriptase (hTERT) mRNA, hTERT protein and reduction in hTERT telomerase activity. CDDO-Me inhibited multiple transcription factors that regulate hTERT expression positively (Sp1, c-Myc and NF-κB) and negatively (CTCF, E2F-1 and MAD1). CDDO-Me inhibited protein levels of DNA methyl transferases DNMT1 and DNMT3a, which also resulted in hypomethylation of hTERT promoter. In addition, transcriptionally active chromatin markers, such as acetylated histone H3 (Lys 9), acetylated histone H4, di-methyl H3 (Lys 4) and tri-methyl H3 (Lys 9) were all reduced in pancreatic cancer cells treated with CDDO-Me. Chromatin immunoprecipitation analysis showed decreased histone deacetylation and histone demethylation at hTERT promoter. Collectively, these results indicate that down-regulation of telomerase through epigenetic mechanisms plays a critical role in induction of apoptosis in pancreatic cancer cells by CDDO-Me.

MART-10, a novel vitamin D analog, inhibits head and neck squamous carcinoma cells growth through cell cycle arrest at G0/G1 with upregulation of p21 and p27 and downregulation of telomerase

For the head and neck squamous cell carcinoma (HNSCC), surgery in combination with radiation therapy is the current standard treatment. However, the complex anatomy and important functions over the head and neck region often make HNSCC patients with severe comorbidities. Even after aggressive treatment, the 5year survival for HNSCC patients is only around 61%. Thus, new therapeutic regimens against HNSCC are urgently needed. 1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3] is a potent anti-tumor agent in a variety of pre-clinical studies, but its clinical application is impeded by hypercalcemic side effect. A new class of less-calcemic 1α,25(OH)2D3 analog, MART-10 (19-nor-2α-(3-hydroxypropyl)- 1α,25-Dihydroxyvitamin D3), has been shown to be much more potent than 1α,25(OH)2D3 in inhibiting cancer cell growth in vitro and in vivo without inducing hypercalcemia. In this study, we compared the antiproliferative activity of MART-10 with 1α,25(OH)2D3 and the mechanism responsible for the inhibition in FaDu and SCC-25 squamous carcinoma cells. Our results demonstrate that MART-10 is more potent than 1α,25(OH)2D3 in suppressing FaDu and SCC-25 cell growth through greater cell cycle arrest at G0/G1, accompanied by a greater downregulation of ki-67 expression and upregulation of p21 and p27. We also showed that telomerase expression in SCC-25 was suppressed to a greater extent by MART-10 than by 1α,25(OH)2D3. Thus, given the previously-proven in vivo antitumor effect and safety of MART-10 and bleak background of HNSCC, based on our current result, we concluded that MART-10 has a potential as a chemo-preventive and – therapeutic agent to treat HNSCC.

Telomerase downregulation induces proapoptotic genes expression and initializes breast cancer cells apoptosis followed by DNA fragmentation in a cell type dependent manner

The aim of the study was to analyze the consequence of silencing genes coding for the key subunits of the telomerase complex, i.e. TERT, TERC and TP1 in human breast cancer MCF7 and MDA-MB-231cells. The transfection was performed using Lipofectamine2000 and pooled siRNAs. The cytotoxic and/or antiproliferative effect of siRNA was measured by the SRB assay, the cell cycle was analysed by flow cytometry and DNA fragmentation by TUNEL analysis. Telomerase activity was assessed by TRAP, followed by PAGE and ELISA assays. Telomerase downregulation was also assessed using qPCR in order to estimate the changes in the expression profile of genes engaged in apoptosis. It was revealed that treatment of breast cancer cells with different siRNAs (100 nM) resulted in a cell type and time-dependent effects. The downregulation of telomerase subunits was followed by reduction of telomerase activity down to almost 60 % compared to control cells. However, a significant effect was only observed when the TERT subunit was downregulated. Its silencing resulted in a significant (p < 0.05) increase of apoptosis (over 10 % in MCF7 and about 5 % in MDA-MB-231 cells, corresponding to the Annexin V assay) and DNA fragmentation (almost 30 % in MCF7 and over 25 % in MDA-MB-231 cells). Interestingly, also several proapoptotic genes were induced after the downregulation of the key telomerase subunit, including Bax, Bik or caspase-1 and caspase-14, as well as NGFR and TNFSF10 which were upregulated twice and more.