Telomerase Activity Of MCF-7 Cells Research Articles

The effects of Hericium erinaceus extracts on cell viability and telomerase activity in MCF-7 cells

Abstract Objectives This study aims to determine the effects of Hericium erinaceus extracts on cell viability and the effects of H. erinaceus water extract on the telomerase activity of MCF-7 cells. H. erinaceus is an edible mushroom widely used in traditional Chinese medicine. Although its various therapeutic properties, the literature has not yet submitted evidence about H. erinaceus for its effects on the telomerase activity of MCF-7 cells. Methods MCF-7 cells were treated with ethanol, ethanol-water, ether, ethyl acetate, methanol-water, and water extracts to determine the effects on cell viability using the WST8 method. The TeloTAGGG Telomerase PCR ELISA kit was used to assess telomerase activity. Results The water extract was determined to be the most efficient extract to decrease cell viability. The water extract’s half-maximal inhibitory concentration was 250 μg/mL at 72 h. It is found that H. erinaceus has no statistically significant effect compared to positive control on reducing telomerase activity. We found a statistically significant difference in telomerase activity % between H. erinaceus water extracts and negative control (p<0.05). Conclusions Consequently, these differences in telomerase activity are a significant association rather than inferring action. It is considered that water extract shows its cell viability inhibition effects through different mechanisms.

Bilateral telomerase-associated impacts of Spirulina platensis extracts: Activation versus inhibition

IntroductionTelomerase is an enzyme responsible for regulating the length of telomeres in eukaryote chromosomes. Activation and inhibition of telomerase results in the extension and shortening of telomeres, respectively. The mentioned phenomena are attributed to anti-aging and anticancer mechanisms. Spirulina platensis extract has attracted a great deal of attention, as an anti-aging factor. MethodsThe regulatory impacts of different extracts of these microalgae on telomerase activity in MCF-7 and HDF cells were investigated using a Telomeric Repeat Amplification Protocol and ELISA, in vitro. ResultsThe methanolic extract of S. platensis increased the telomerase activity by 139% and simultaneously showed high toxicity on the MCF-7 cells (82%) with IC50=0.035 mg/ml. Despite this, the methanolic extract demonstrated an unstable activity of HDF cells. In contrast to the methanolic extract, the aqueous extract of S. platensis toxified the cancer cells at the highest rate of 77% (IC50=0.98 mg/ml); however, it inhibited the telomerase activity of MCF-7 cells up to 52%. Additionally, the aqueous extract acted as an anti-aging factor on the HDF cells through upregulating the telomerase activity by 84% at 0.004 mg/ml. The present research suggests that different extracts of S. platensis behave variously on different cell types. Except the aqueous extract, other extracts of S. platensis are capable of inhibiting MCF-7 cancer cells based on a mechanism different than telomerase inhibition. ConclusionThe aqueous extract of S. platensis seems to be a safe alternative for use as an anti-aging factor as well as an anticancer agent.

Identification of an RNA aptamer binding hTERT-derived peptide and inhibiting telomerase activity in MCF7 cells.

Human telomerase reverse transcriptase is an essential rate-limiting component of telomerase complex. hTERT protein in association with other proteins and the human telomerase RNA (hTR) shows telomerase activity, essential for maintaining genomic integrity in proliferating cells. hTERT binds hTR through a decapeptide located in the RID2 (RNA interactive domain 2) domain of N-terminal region. Since hTERT is essential for telomerase activity, inhibitors of hTERT are of great interest as potential anti-cancer agent. We have selected RNA aptamers against a synthetic peptide from the RID2 domain of hTERT by employing in vitro selection protocol (SELEX). The selected RNAs could bind the free peptide, as CD spectra suggested conformational change in aptamer upon RID2 binding. Extracts of cultured breast cancer cells (MCF7) expressing this aptamer showed lower telomerase activity as estimated by TRAP assay. hTERT-binding RNA aptamers hold promise as probable anti-cancer therapeutic agent.

The putative tumor suppressor C53 interacts with the human telomerase reverse transcriptase hTERT and regulates telomerase activity

Telomerase is a large ribonucleoprotein complex that contains a catalytic telomerase reverse transcriptase (TERT) and an RNA template. Telomerase activity is tightly controlled by TERT expression, which is regulated at both the transcriptional and post-translational levels. However, the detailed molecular mechanisms of telomerase regulation and function are not fully understood. To identify cofactors that contribute to telomerase regulation, we employed a yeast two-hybrid system to screen for hTERT-interacting proteins, using the hTERT T-motif as bait. We identify C53 as a novel hTERT interaction partner. We show that C53 interacts with hTERT both in vivo and in vitro. C53 depletion increases telomerase activity, and C53 overexpression inhibits telomerase activity in MCF7 cells. In addition, the C53 leucine zipper domain (amino acids 301–400) is required for interaction with hTERT. Deleting the leucine zipper domain eliminates C53 interaction with hTERT and abrogates its inhibitory effect on telomerase activity. Taken together, our results demonstrate that C53 is a novel hTERT-associated protein that negatively regulates telomerase activity.

The β-carboline alkaloid harmine inhibits telomerase activity of MCF-7 cells by down-regulating hTERT mRNA expression accompanied by an accelerated senescent phenotype

The end replication problem, which occurs in normal somatic cells inducing replicative senescence, is solved in most cancer cells by activating telomerase. The activity of telomerase is highly associated with carcinogenesis which makes the enzyme an attractive biomarker in cancer diagnosis and treatment. The indole alkaloid harmine has multiple pharmacological properties including DNA intercalation which can lead to frame shift mutations. In this study, harmine was applied to human breast cancer MCF-7 cells. Its activity towards telomerase was analyzed by utilizing the telomeric repeat amplification protocol (TRAP). Our data indicate that harmine exhibits a pronounced cytotoxicity and induces an anti-proliferation state in MCF-7 cells which is accompanied by a significant inhibition of telomerase activity and an induction of an accelerated senescence phenotype by over-expressing elements of the p53/p21 pathway.

Stable expression of promyelocytic leukaemia (PML) protein in telomerase positive MCF7 cells results in alternative lengthening of telomeres phenotype

BackgroundCancer cells can employ telomerase or the alternative lengthening of telomeres (ALT) pathway for telomere maintenance. Cancer cells that use the ALT pathway exhibit distinct phenotypes such as heterogeneous telomeres and specialised Promyelocytic leukaemia (PML) nuclear foci called APBs. In our study, we used wild-type PML and a PML mutant, in which the coiled-coil domain is deleted (PML C/C-), to investigate how these proteins can affect telomere maintenance pathways in cancer cells that use either the telomerase or ALT pathway.ResultsStable over-expression of both types of PML does not affect the telomere maintenance in the ALT cells. We report novel observations in PML over-expressed telomerase-positive MCF7 cells: 1) APBs are detected in telomerase-positive MCF7 cells following over-expression of wild-type PML and 2) rapid telomere elongation is observed in MCF7 cells that stably express either wild-type PML or PML C/C-. We also show that the telomerase activity in MCF7 cells can be affected depending on the type of PML protein over-expressed.ConclusionOur data suggests that APBs might not be essential for the ALT pathway as MCF7 cells that do not contain APBs exhibit long telomeres. We propose that wild-type PML can either definitively dominate over telomerase or enhance the activity of telomerase, and PML C/C- can allow for the co-existence of both telomerase and ALT pathways. Our findings add another dimension in the study of telomere maintenance as the expression of PML alone (wild-type or otherwise) is able to change the dynamics of the telomerase pathway.

Tetrasubstituted naphthalene diimide ligands with selectivity for telomeric G-quadruplexes and cancer cells

A series of tetrasubstituted naphthalene diimide compounds with N-methylpiperazine end groups has been synthesized and evaluated as G-quadruplex ligands. They have high affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA. CD studies show that they induce formation of a parallel G-quadruplex topology. They inhibit the binding of hPOT1 and topoisomerase IIIα to telomeric DNA and inhibit telomerase activity in MCF7 cells. The compounds have potent activity in a panel of cancer cell lines, with typical IC50 values of ∼0.1μM, and up to 100-fold lower toxicity in a normal human fibroblast cell line.

Effect of c-myc antisense oligonucleotide on telomerase activity in MCF-7 cells

目的 研究脂质体LipfectAMINETM(LR)介导的c-myc反义寡核苷酸(ASODN)对MCF-7细胞端粒酶活性的影响及其机制.方法 端粒重复序列扩增聚丙烯酰胺凝胶电泳(TRAP-PAGE)及端粒重复序列扩增酶联免疫吸附测定(TRAP-ELISA)定量检测不同时间段MCF-7细胞的端粒酶活性,流式细胞仪检测c-myc蛋白表达.结果 TRAP-PAGE结果显示,c-myc ASODN对MCF-7细胞的作用,随时间的递增(24 h、48 h、72 h),端粒酶梯度条带明显地减少,端粒酶活性明显降低.TRAP-ELISA显示,2.5μmol/L的c-myc ASODN作用MCF-7细胞48 h吸光度A值降为0.486.4±0.041,作用72 h A值降为0.263±0.034,与未经任何处理的MCF-7细胞A值0.684±0.031相比,端粒酶活性明显受到抑制,分别与正义寡核苷酸组、空白对照组相比差异有统计学意义(P＜0.05).未经任何处理的MCF-7细胞c-myc蛋白阳性率为(99.684±2.937)%,经c-myc ASODN作用48h c-myc蛋白阳性率低至(61.295±2.825)%,c-myc ASODN作用72 h c-myc蛋白阳性率低至(29.482±2.726)%.而c-myc正义寡核苷酸(SODN)无上述作用.结论 c-myc ASODN能有效降低MCF-7细胞端粒酶活性和c-myc蛋白表达。

Attenuation of telomerase activity by hammerhead ribozyme targeting human telomerase RNA induces growth retardation and apoptosis in human breast tumor cells

Ribozyme possesses specific endoribonuclease activity and catalyzes the hydrolysis of specific phosphodiester bonds, which results in the cleavage of target RNA sequences. Here, we evaluated the ability of hammerhead ribozymes targeting human telomerase RNA (hTR) to inhibit the catalytic activity of telomerase and the proliferation of cancer cells. Hammerhead ribozymes were designed against 7 NUX sequences located in open loops of the hTR secondary structure. We verified the ribozyme specificity by in vitro cleavage assay by using a synthetic RNA substrate. Subsequently, we introduced ribozyme expression vector into human breast tumor MCF-7 cells and assessed the biologic effects of ribozyme. Hammerhead ribozyme R1 targeting the template region of hTR efficiently cleaved hTR in vitro, and stable transfectants of this ribozyme induced the degradation of target hTR RNA and attenuated telomerase activity in MCF-7 cells. Moreover, the ribozyme R1 transfectant displayed a significant telomere shortening and a lower proliferation rate than parental cells. Clones with reduced proliferation capacity showed enlarged senescence-like shapes or highly differentiated dendritic morphologies of apoptosis. In conclusion, the inhibition of telomerase activity by hammerhead ribozyme targeting the template region of the hTR presents a promising strategy for inhibiting the growth of human breast cancer cells.

Effect of estrogen on telomerase activity in human breast cancer cells.

To investigate the effects of estrogen (E2) on telomerase activity and its mechanism in human breast cancer cells, estrogen receptor positive MCF-7 cells were treated with different concentrations of E2. Telomerase activity was measured by using TRAP-ELISA method, the cell cycle phases analyzed by using flow cytometry, and the expression of Cyclin D1 detected by using immunohistochemistry method. The results showed that telomerase activity levels were increased in MCF-7 cells treated with 10(-8) mol/L E2 during the observed period (P < 0.05), and E2 increased telomerase activity levels in a dose-dependent manner(10(-10)-10(-8) mol/L); Simultaneously, the cell cycle phases of MCF-7 cells treated with 10(-8) mol/L E2 were changed significantly: G0/G1 phase decreased from 60.52% to 50.93%. S phase increased from 29.03% to 30.83%; However, the expression of Cyclin D1 was decreased. It was concluded that estrogen can upregulate telomerase activity of MCF-7 cells, and the effect can be blocked by antiestrogen tamoxifen. Its mechanism may be closely associated with modulation of cell cycle phases.

Curcumin inhibits telomerase activity through human telomerase reverse transcritpase in MCF-7 breast cancer cell line

The inhibitory effect of curcumin, the yellow-colored pigment from turmeric, on telomerase activity was analyzed in human mammary epithelial (MCF-10A) and breast cancer (MCF-7) cells. Telomerase activity in MCF-7 cells is 6.9-fold higher than that of human mammary epithelial cells. In MCF-7 cells, telomerase activity decreased with increasing concentrations of curcumin, inhibiting about 93.4% activity at 100μM concentration. The inhibition of telomerase activity in MCF-7 cells may be due to down-regulation of hTERT expression. Increasing concentrations of curcumin caused a steady decrease in the level of hTERT mRNA in MCF-7 cells whereas the level of hTER and c-myc mRNAs remained the same. Our results suggest that curcumin inhibits telomerase activity by down-regulating hTERT expression in breast cancer cells and this down-regulation is not through the c-myc pathway.

Inhibition of cell growth and telomerase activity of breast cancer cells in vitro by retinoic acids.

The effects of retinoic acid (RA) and its analogs, all-trans RA, 9-cis RA and 13-cis RA, were investigated in human breast cancer MCF-7 cells and immortalized breast epithelial cell line MCF-10A. RA inhibited the telomerase activity of MCF-7 cells in a wide range of concentrations. RA at 10 microM also inhibited the growth of MCF-7 cells in a time-dependent manner. However, no significant growth inhibition was found between untreated control and RA-treated MCF-10A cells. Moreover, a marked inhibition of telomerase activity by RA was detected early in MCF-7 cells (after 24 h of RA treatment), which was preceded by a reduction of hTERT mRNA expression (after 12 h of RA treatment). However, MCF-10A cells showed a reduction of telomerase activity and down-regulation of hTERT after 4 days of RA treatment. Simultaneous changes in hTERT mRNA expression and telomerase activity were found for MCF-10A cells. The expressions of hTR and hTEP1 telomerase component genes were not changed after RA treatment. These results indicate that the anti-breast cancer activity of RA could be mediated by its ability to down-regulate the expression of hTERT telomerase gene.

A human breast cancer model for the study of telomerase inhibitors based on a new biotinylated-primer extension assay.

Telomerase is an RNA-dependent polymerase that synthesizes telomeric DNA (TTAGGG)n repeats. The overall goal of our work was to establish human cancer models that can be used to design clinical trials with telomerase inhibitors. The objectives of this study were (1) to set up a human breast cancer system that allows evaluation of the effects of telomerase inhibitors in cultured cells using a non-amplified telomerase assay and (2) to test this system using two drugs (cisplatin and TMPyP4) that affect the telomerase expression in breast cancer cells in culture. We first compared the telomerase activity in a variety of human breast cancer cell lines to that of other tumour types using a new biotinylated-primer extension assay. Our method, based on a non-amplified primer extension assay shows the direct incorporation of 32P-labelled nucleotides induced by telomerase on human telomeric primers. The 32P-dGTP labelled telomerase-extended 5′-biotinylated (TTAGGG)3 primer can subsequently be separated using streptavidin-coated magnetic beads. As compared to other non-amplified method, we showed that this procedure improved the characterization and the quantification of the banding pattern resulting from telomerase extension by reducing the radioactive background. Using this method, we observed that telomerase activity varies markedly in a panel of 39 human cancer cell lines. For example, MCF7 breast cancer cells in culture showed intermediate telomerase activity corresponding to 33.8 ± 3.4% of that of the HeLa cells (reference cell line). Similarly, the telomere length varied with each cell line (average: 6.24 ± 6.16). No correlation between the level of telomerase and telomere length was observed, suggesting that a high processivity is not required to maintain telomeres and that, in some cell lines, another mechanism of telomere elongation can maintain telomere length. From this study, we selected MCF7 and MX1 models that showed reproducible telomerase activity and a relatively limited telomere length for the testing of potential telomere–telomerase interacting agents. Using cisplatin and a new porphyrin-derived compound TMPyP4, we showed that our model was able to detect a down-regulation of the telomerase activity in MCF7 cells in culture and in a human MX1 tumour xenografts. Based on these results, a breast cancer model for evaluating telomerase and telomere interactive agents is proposed. © 1999 Cancer Research Campaign