Identification of a series of potent telomerase inhibitors using a time-resolved fluorescence-based assay

A high-throughput, nonradioactive telomerase assay using a europium-labeled oligonucleotide probe and time-resolved fluorescence has been developed to detect PCR-amplified telomerase products. The use of a thermally activated TAQ polymerase, rather than a wax barrier, to implement a PCR hot-start facilitated the use of laboratory automation equipment. Results obtained with this high-throughput protocol correlate well with those obtained with the TRAP protocol. Screening a set of 125,000 compounds from the Berlex library, we identified a set of isothiazolone-containing telomerase inhibitors. The most potent of these inhibitors have submicromolar IC₅₀ values and may be reacting with a telomerase thiol. These compounds may be useful tools to evaluate the effects of telomerase in cancer cells. Drug Dev. Res. 43:109–116, 1998. © 1998 Wiley-Liss, Inc.     

SYBR Green实时定量端粒重复序列扩增法检测端粒酶活性

【摘要】目的 采用实时定量端粒重复序列扩增法( RQ- TRAP法)检测不同细胞端粒酶活性。方法 用RQ- TRAP和TRAP-ELISA两种方法同时检测12种细胞的端粒酶活性性,并比较两种方法的检测结果。结果 RQ-TRAP方法能准确特异地检测系列稀释的293T细胞蛋白提取液的端粒酶活性,灵敏度可达8个细胞,扩增效率为99%。阴性对照组则未检测到端粒酶活性。RQ-TRAP方法测得12个细胞系中端粒酶的活性与TRAP-ELISA方法结果有相关性(R2=0.7625)。结论 RQ-TRAP方法检测端粒酶可行,与TRAP-ELISA方法相比,RQ-TRAP方法具有成本低,减少时长和支持高通量等优点,是一种新的可快速可靠定量端粒酶活性的方法。      

高三尖杉酯碱对HL-60细胞端粒酶量的抑制效应

目的　研究高三尖杉酯碱 (HHT)对HL 6 0细胞端粒酶活性的影响及诱导凋亡作用。方法　采用端粒重复序列扩增法 (TRAP) 酶联免疫吸附试验(ELISA)检测了HL 6 0细胞的端粒酶量变化 ,用细胞形态学观察 ,DNA琼脂糖凝胶电泳 ,流式细胞术检测和DNA片段原位末端标记 (TUNEL)法检测了细胞凋亡现象。结果　 5～ 5 0 0 μg·L- 1HHT处理HL 6 0细胞 0～ 4 8h ,HL 6 0细胞端粒酶量呈剂量依赖性和时间依赖性下降。同时 ,HL 6 0细胞发生了凋亡。结论　HHT有降低HL 6 0细胞的端粒酶量 ,诱导细胞凋亡作用。     

Synergy Between 3′Azido-3′deoxythymidine and Paclitaxel in Human Pharynx FaDu Cells

Purpose. We recently demonstrated simultaneous targeting of telomere and telomerase as a novel cancer therapeutic approach, and that telomerase inhibitors such as 3azido-3deoxythymidine (AZT) significantly enhanced the antitumor activity of paclitaxel, which causes telomere erosion, in telomerase-positive human pharynx FaDu tumors in vitro and in vivo (1). The present study evaluated the synergy between AZT and paclitaxel to identify optimal combinations for future clinical evaluation. Methods. FaDu cells were incubated with or without AZT for 24 h and then treated with AZT with or without paclitaxel for an additional 48 h. Under these conditions, single agent paclitaxel produced a 60% maximum reduction of cell number (IC₅₀ was 7.3 nM), and single agent AZT produced a 97% reduction (IC₅₀ was 5.6 M). Synergy was evaluated using fixed-concentration and fixed-ratio methods, and data were analyzed by the combination index method. Results. The results indicate a concentration-dependent synergy between the two drugs; the synergy was higher for combinations containing greater paclitaxel-to-AZT concentration ratios and increased with the level of drug effect. For example, in combinations containing 1 M AZT, synergy was 1.3-fold at the 20% effect level and 3.1-fold at the 60% effect level. Because the major antitumor activity, determined by comparing the posttreatment cell number to the pretreatment cell number, was antiproliferation at the 20% effect level and cell kill at the 60% effect level, our results suggest that AZT mainly enhances the cell kill effect of paclitaxel. Conclusion. In summary, the present study demonstrates a synergistic interaction between paclitaxel and AZT and supports a combination using a low and nontoxic AZT dose in combination with a therapeutically active dose of paclitaxel.     

克痒舒洗液联合双唑泰栓治疗外阴阴道假丝酵母菌病的临床研究

目的 对当归不同炮制品中大肠埃希菌变化进行定量分析。方法 建立荧光定量PCR方法,对当归不同炮制品、不同产地、不同收集企业、不同储藏时间的大肠埃希菌进行定量分析。结果 不同炮制品中大肠埃希菌数量变化为生当归 > 土炒当归 > 酒当归;在不同产地的当归中以甘肃岷县地区所含的大肠埃希菌的数量最少;与零售企业相比,生产销售企业的当归和酒当归中大肠埃希菌的数量较少;不同储藏时间对当归和酒当归中大肠埃希菌数量有一定影响,随储藏时间的增加,大肠埃希菌的数量也在增加。对部分代表性样品进行平板计数法与荧光定量PCR法比较时发现,平板计数法结果多呈阴性,荧光定量PCR结果均呈阳性。结论 所建立的荧光定量PCR法特异性、灵敏度、可靠性以及报告周期均优于平板计数法,可为当归不同炮制品中大肠埃希菌的快速、准确定量检测提供有效手段。     

大蹼铃蟾蛋白提取物对膀胱癌细胞株的抑制作用及对端粒酶的影响

目的　探讨大蹼铃蟾蛋白提取物对膀胱癌细胞株的抑制作用及对端粒酶的影响。方法　采用肿瘤细胞培养方法 ,检测大蹼铃蟾蛋白提取物对膀胱癌细胞株T2 4、BIU 87、ScaBer的抑制作用 ;以TRAP PCR ELISA方法检测大蹼铃蟾蛋白提取物对膀胱癌细胞株端粒酶表达的影响。结果　大蹼铃蟾蛋白提取物表现出对膀胱癌细胞株有较强的抑制作用 ;对膀胱癌细胞株端粒酶表达则无影响。结论　大蹼铃蟾蛋白提取物对多个膀胱癌细胞株具有直接细胞杀伤作用 ,其作用机制不通过抑制端粒酶活性     

A new telomerase inhibitor and apoptosis‐inducing agent in leukemia: perylene derivative G‐quadruplex ligand Tel03

Tel03 (N,N′‐Bis‐(2‐(dimethylamino)ethyl)‐3,4,9,10‐perylentetra‐carboxylic acid diimide), a perylene derivative, was synthesized and its interaction with different forms of human telomeric DNA examined . The effect of Tel03 on leukemia cells in short‐term cultures was also used to explore its molecular actions. Electrospray ionization mass spectrometry (ESI‐MS) analysis indicated that Tel03 specifically recognizes telomeric quadruplex, not duplex DNA. After the treatment with Tel03 (0.1–10 µM) for 48 h, telomerase activity in K562 leukemia cell extracts was inhibited in a dose‐dependent manner as assessed with a telomere repeat amplification protocol (TRAP) assay. MTT assay results showed that Tel03 (0.1–10 µM) inhibited the proliferation of K562 cells after treatment for 72 h. In addition, apoptosis was observed. Using real‐time quantitative PCR and Western blot analysis, the expression of c‐myc and bcl‐2 was markedly down‐regulated at both the mRNA and protein levels. However, no changes were observed in the expression of bax. We conclude that G‐quadruplex ligand, Tel03, is a novel telomerase inhibitor and apoptosis‐inducing agent in leukemia cells acting via down‐regulation of bcl‐2 and c‐myc. Tel03 has the potential to be developed as an antileukemia drug. Drug Dev Res 69:235–241, 2008. © 2008 Wiley‐Liss, Inc.     

Telomere Amount and Length Assay

Purpose. Telomeres are specific DNA structure at the ends of chromosomes to protect chromosomes from fusion, recombination, and degradation. Telomere length changes are implicated in cell senescence, aging, tumorigenesis, and DNA repair. The standard method for measuring telomere length is Southern blot analysis. This method has several disadvantages, i.e., loss of DNA during membrane blotting, high background due to nonspecific binding of telomere probe to membrane, and loss of telomeric signal due to extensive washing. These limitations resulted in a low signal-to-noise ratio and, therefore, reduced sensitivity and reproducibility. The multi-step Southern blot is also highly labor-intensive. The present study was to develop a more quantitative assay of telomeric amount and length (TALA). Methods. TALA was based on solution hybridization and did not require blotting, prehybridization, and washing. The major steps were (a) DNA preparation and digestion with restriction endonucleases, (b) hybridization between DNA and telomeric probe, (c) agarose gel electrophoresis, and (d) autoradiography and data analysis. Results. The telomere amount measured by TALA was linearly correlated with the amount of DNA analyzed (r² = 0.985, P < 0.01). The telomere length measured by TALA also correlated with the telomere length determined by fluorescence in situ hybridization (r² = 0.99, P < 0.01). Compared to the Southern blot analysis, TALA showed a 4-fold greater sensitivity, 4.6-fold higher signal-to-noise ratio, >2 fold-higher reproducibility, and 4-fold less time requirement. Conclusion. We report here a rapid, sensitive, and quantitative assay for measuring telomere length and amount.     

Telomerase inhibitors: a patent review (2010–2015)

Introduction: Telomerase is a ribonucleoprotein that catalyses the addition of telomeric repeat sequences (having the sequence 5′-TTAGGG-3′ in humans) to the ends of chromosomes. Telomerase activity is detected in most types of human tumours, but it is almost undetectable in normal somatic cells. Therefore, telomerase is a promising therapeutic target. To date, the known inhibitors of telomerase include nucleoside analogues, oligonucleotides and G-quadruplex stabilizers. This review highlights recent advances in our understanding of telomerase inhibitors, the relationships between telomerase inhibitors, cancer, and fields such as inflammation.

Areas covered: This review summarizes new patents published on telomerase inhibitors from 2010 to 2015.

Expert opinion: The review provides a brief account of the background, development, and on-going issues involving telomerase inhibitors. In particular, this review emphasizes imetelstat (GRN163L) and some typical G-quadruplex stabilizers that participate in telomerase inhibition. Overall, the research scope of antineoplastic is becoming broader and telomerase inhibitors have been shown to be a promising therapeutic target. Therefore, novel antineoplastic agents with greater activity and higher specificity must be developed.     

The mechanisms of action of Tianhua™ on antitumor activity in lung cancer cells

Context: Tianhua (TH-R) is extracted from Trichosanthes kirilowii Maxim (Cucurbitaceae) containing trichosanthin, a traditional Chinese medicine, which has been locally reported to have good anticancer effects in vivo in both animal and human models. However, there have been several reports that trichosanthin has an anticancer effect involving apoptosis.

Objective: To investigate other anticancer effects of TH-R, various tumorigenesis parameters were verified.

Materials and methods: Telomerase activity, anti-apoptosis, anti-migration and immunomodulatory activity were estimated by telomeric repeat amplification protocol assay (TRAP), flow cytometry, Boyden chamber assay and ELISA assay, respectively.

Results: In our studies, we are the first to find that TH-R had a cytotoxic effect on lung cancer cells in MTS assays; it could change the cell cycle distribution of human lung cancer cells (A549 cell line) and induce apoptosis. Further anti-telomerase effects in human lung adenocarcinoma A549 cells using the TRAP assay were noted. TH-R also had an aggregation effect on peripheral blood lymphocytes, but no effect on stimulating peripheral lymphocytes to produce human interferon-γ(IFN-γ). TH-R could inhibit the migration, or metastatic ability, of A549 cells by Boyden chamber assay. In the oral feeding therapy of an in vivo mouse model, there was an initial inhibition of A549 cancer cell growth, but no statistical difference after one month of therapy.

Discussion and conclusion: It has been proven that medicinal herbs such as Tianhua have positive effects against cancer through preventing or inhibiting the process of lung tumorigenesis.     

荷包牡丹碱对人肺癌A549细胞生长及端粒酶活性的抑制作用

目的探讨荷包牡丹碱对人肺癌A549细胞生长的抑制作用及其作用机制。方法 A549细胞加入荷包牡丹碱0～200μmol.L-1分别作用24,48和72 h,MTT法测定A549细胞的生长抑制作用。荷包牡丹碱0～20μmol.L-1作用72 h,端粒重复序列扩增(TRAP)法测定端粒酶活性。变温紫外法检测荷包牡丹碱9μmol.L-1对端粒酶G-四链体的稳定作用。结果荷包牡丹碱25,50,100和200μmol.L-1作用细胞72 h后的抑制率分别为33.4%,88.2%,88.6%和89.4%,明显高于正常对照组细胞(P<0.05),并具有量效(r=0.906,P<0.05)和时效(r=0.949,P<0.05)性。与正常对照组相比,荷包牡丹碱5,10,15和25μmol.L-1可有效抑制A549细胞端粒酶的活性(P<0.05),相对TRAP端粒酶活性从正常对照组的1.471±0.102分别降低为1.093±0.054,1.013±0.016,0.554±0.034,0.365±0.081(P<0.05)。荷包牡丹碱9μmol.L-1使G-四链体的熔点值从正常对照组的48℃提高到54℃。结论荷包牡丹碱可以通过稳定G-四链体结构,抑制端粒酶活性,有效抑制人肺腺癌细胞A549的生长。     

A Novel Topoisomerase II Poison GL331 Preferentially Induces DNA Cleavage at (C/G)T Sites and Can Cause Telomere DNA Damage

Purpose. Topoisomerase II (Topo II) preferentially cuts DNA at alternating purine-pyrimidine repeats. Different Topo II poisons may affect Topo II to produce distinct drug-specific DNA cleavage patterns. GL331 is a new podophyllotoxin derivative exhibiting potent Topo II-poisoning activity. Therefore, the sequence selectivity of GL331-induced DNA cleavage was determined. Methods. Human gastric adenocarcinoma SC-M1 cells were treated with GL331, and the resultant DNA fragments were isolated by SDS-K⁺ precipitation. These DNA fragments were further cloned and sequenced to exhibit GL331-induced DNA cleavage sites. In addition, the telomere damage was detected by Southern blot analyses using a (TTAGGG)₄ probe. GL331's effect on telomerase was examined using the TRAP assay. Results. The selective sequences of GL331-induced DNA cleavage were analyzed. The first nucleotide 3-terminal to the cleavage sites was preferentially C or G and followed by the second nucleotide T. More than 50% of GL331-induced DNA cleavage fragments exhibited AT-rich sequences in the first 20 nucleotides. In addition, the telomeric damage was observed both from GL331-treated SC-M1 cells and in vitro incubation of genomic DNA with GL331 and purified human Topo II. Although GL331 treatment reduced cellular telomerase activity, in vitro reaction data suggested that GL331 was not a telomerase inhibitor. Conclusions. GL331 preferentially induced Topo II-mediated DNA cleavage at (C/G)T sites. Because the telomeric repeat sequence contains GL331's GT preference site, the telomere was identified as one of the targets of GL331-induced DNA damage.     

洋葱伯克霍尔德菌群3种检测方法的比较

目的 通过对环介导等温扩增(loop-mediated isothermal amplification,LAMP)、基于聚合酶链式扩增(polymerase chain reaction,PCR)的SYTO 9染料法及TaqMan探针实时荧光定量PCR法(简称TaqMan探针法)3种检测方法的比较,旨在建立一种能够快速、准确检测24株洋葱伯克霍尔德菌群的PCR方法。方法 根据 NCBI 数据库中24株洋葱伯克霍尔德菌的分子生物学信息,筛选出洋葱伯克霍尔德菌所特有的多个候选序列片段,设计能同时检出24株洋葱伯克霍尔德菌的特异性引物和探针,同时探索了LAMP法、基于PCR的SYTO 9染料法及Taqman探针法3种检测方法,优化筛选出最佳退火温度,并采用39株实验菌株对洋葱伯克霍尔德菌群检测方法开展了验证研究。结果 采用LAMP法无法实现对洋葱伯克霍尔德菌群的有效检出,采用SYTO 9染料法和TaqMan探针法可以实现对20多株洋葱伯克霍尔德菌群的有效检出,而TaqMan探针法扩增效率更高,检测灵敏度、重复性、稳定性更好,能够满足本研究的要求。结论 本研究建立了洋葱伯克霍尔德菌群的TaqMan探针快速检测方法,相较于LAMP法和SYTO 9染料法,该方法具有快速、简便和敏感性高等优点,为洋葱伯克霍尔德菌群的快速检测提供了技术支持。     

荧光定量PCR法测定地特胰岛素原料中酿酒酵母宿主DNA残留量

目的建立检测地特胰岛素原料中宿主DNA残留量的荧光定量核酸扩增(Real-time PCR)方法并进行验证,用于该产品的质量控制。方法选择酿酒酵母5s RNA作为靶基因设计引物,提取地特胰岛素原料中的残留宿主DNA,采用Real-time PCR SYBRGreen染料法对标准DNA和样品进行测定,绘制标准曲线并分析样品中的DNA残留量。对建立的方法进行方法学验证,并测定3批地特胰岛素原料中的残留宿主DNA。结果酿酒酵母基因组DNA质量浓度在0.18～180000ng/mL线性良好(r~2=0.998 5);回收率均在80.0%～106.3%,检测3批地特胰岛素原料的宿主DNA残留量均低于进口药品注册标准限度。结论该方法可用于酿酒酵母生产的地特胰岛素原料中残留宿主DNA的定量测定。     

Angelica sinensis polysaccharides inhibit endothelial progenitor cell senescence through the reduction of oxidative stress and activation of the Akt/hTERT pathway

Abstract

Context: Angelica sinensis (Oliv.) Diels (Apiaceae) polysaccharides (ASP) may play a key role in anti-ischemic activity. However, the anti-atherosclerotic activity and mechanism are unknown.

Objective: This study investigated the protective effects of ASP against ox-LDL-induced senescence of EPCs and explored its underlying molecular mechanisms.

Materials and methods: Mononuclear cells were isolated from bone marrow (BM) of SD rats and differentiated to EPCs. EPCs were exposed to oxidized low-density lipoprotein (ox-LDL, 10?µg/mL, 24?h) and incubated with or without high-dose (100?µg/mL, 48?h) or low-dose (20?µg/mL, 48?h) ASP. Another group of EPCs was pre-treated with Wortmannin (100?nM, 45?min), a PI3K/Akt inhibitor. EPC senescence, telomerase activity, and superoxide anion levels were assessed using SA-β-galactosidase staining, telomerase PCR-ELISA analysis, and DHE staining, respectively. The expression of related proteins, including Akt, p-Akt, hTERT, p-hTERT, and gp91phox, were detected using western blot.

Results: EPCs (47.3%) were SA-β-gal positive after treatment by ox-LDL, additionally, ox-LDL significantly increased superoxide anion levels (375% versus 100%), and inhibited telomerase activity (42% versus 100%). However, the pro-senescent effect of ox-LDL was attenuated about three-fold (16.7%), superoxide anion levels were decreased more than two-fold (148%), and telomerase activity was recovered partly (88% versus 42%) in the EPCs when treated with ASP (100?µg/mL). The immunoblotting confirmed that ASP attenuated inhibition of phosphorylation of Akt and hTERT induced by ox-LDL and down-regulated increased the expression of gp91-phox. Moreover, some effects of ASP were partially abrogated in the presence of Wortmannin.

Discussion: Ox-LDL induced senescence of EPCs via inhibition of telomerase activity, which was influenced by oxidative stress and the Akt/hTERT pathway. The inhibition of EPC senescence by ASP could be important for potential therapeutics.

Conclusion: Treatment of EPCs with ASP remarkably attenuates the harmful effects of ox-LDL via augmentation of Akt/hTERT phosphorylation and inhibition of oxidative stress.     

喜树碱诱导人白血病HL-60细胞凋亡过程中端粒酶的调控(英文)

目的:研究在喜树碱诱导人白血病HL—60细胞凋亡过程中端粒酶的调节变化规律.方法:用MTT法测定药物对细胞存活率的影响;用琼脂糖电泳及流式细胞术检测和定量凋亡的发生;用以PCR为基础的TRAP法测定端粒酶活力;逆转录PCR检测凋亡过程中bcl-2及端粒酶亚基hTR、hEST2/hTERT和TLPl/TPl的基因表达水平的变化.结果:端粒酶活力伴随喜树碱诱导HL-60细胞凋亡的发生而逐渐降低,在此过程中端粒酶各亚基的mRNA水平无可见性变化,而bcl-2的基因表达水平则相应下调.结论:端粒酶活力的下调和喜树碱诱导的HL-60凋亡密切相关,端粒酶活力的阻断并非发生在其亚基基因转录水平,bcl-2对端粒酶活力的调节也不是通过影响端粒酶亚基的转录水平来实现的.     

Cyclo-oxygenase-2 (COX-2) mRNA expression correlates with progesterone receptor positivity in human breast cancer

Background: We previously presented evidence showing that cyclo-oxygenase 2 (COX-2) plays an important role in mammary carcinogenesis and angiogenesis in human breast cancer. The present study aims to compare COX-2 mRNA expression with hormone receptor status, S-phase fraction, telomerase activity, and DNA ploidy in human breast cancer.

Methods: Total cellular RNA was extracted from frozen breast tissue samples according to standard methodology. The mRNA copy numbers for COX-2 were determined in 18 infiltrating carcinomas using quantitative RT-PCR and TaqMan methodology. The oestrogen receptor (ER) and progesterone receptor (PgR) status was determined using the ligand-binding technique (ER+?=?>3?fmol/mg, PgR+?=?>5fmol/mg). We also determined DNA ploidy status (diploid or aneuploid), S-phase fraction (<6%?=?low, 6-10%?=?intermediate, > 10%?=?high), and telomerase activity (total protein generated by TRAP assay).

Results: The median COX-2 mRNA copy number per ug of RNA was 126 713 (range?=?15717-2022050). COX-2 expression was significantly associated with PgR positivity (p?=?0.013). The association between COX-2 and DNA diploidy failed to reach a statistical significance (p?=?0.085). No significant association was detected between COX-2 and S-phase fraction, ER status, or telomerase activity.

Conclusions: COX-2 mRNA expression is associated with PgR positivity in human breast cancer. This observation is consistent with the hypothesis that COX-2 upregulates aromatase activity.     

Design and Synthesis of TRAP1 Selective Inhibitors: H-Bonding with Asn171 Residue in TRAP1 Increases Paralog Selectivity

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is overexpressed in the mitochondria of various cancer cells, reprograms cellular metabolism to enable cancer cells to adapt to harsh tumor environments. As inactivation of TRAP1 induces massive apoptosis in cancer cells in vitro and in vivo, the development of TRAP1-selective inhibitors has become an attractive approach. A series of purine-8-one and pyrrolo[2,3-d]pyrimidine derivatives was developed based on TRAP1 structure and identified to be highly selective in vitro for TRAP1 over the paralogous enzymes, Hsp90α and Grp94. The TRAP1-selective inhibition strategy via utilization of the Asn171 residue of the ATP-lid was investigated using X-ray crystallography and molecular dynamics simulation studies. Among various synthesized potent TRAP1 inhibitors, 5f possessed a 65-fold selectivity over Hsp90α and a 13-fold selectivity over Grp94. Additionally, 6f had a half-maximal inhibitory concentration (IC₅₀) of 63.5 nM for TRAP1, with a 78-fold and 30-fold selectivity over Hsp90α and Grp94, respectively.     

Patent Evaluation: A Method for the Detection of Point Mutations

Summary

Novelty: A novel method for the detection of point mutations and genetic variations is described. The method is based on primer extension and the incorporation of detectable nucleoside triphosphates.

Biology: The method for detection of point mutations in sequence variations is dependent on the knowledge of the sequence adjacent to the variable region, preferably immediately toward the 3′ end of the variable nucleotide. By selecting the detection step primers from the region immediately adjacent to the variable nucleotide, this variation can be detected after incorporation of as few as one nucleotide triphosphate. Labelled nucleotide triphosphates matching the variable nucleotide are added and the incorporation of label into the detection step primer is measured. This method, in conjunction with an amplification method such as PCR, can be used with a wide variety of radioactive and non-radioactive detection methods.