OBJECTIVETo study the effects of adramycin to disturb telomeric extention reaction mediated by telomerase of Tca8113 cells by inducing oligonucleotides that contain telomeric repeats to form guanine-quadruplex (G4) structures.

METHODSIn the presence of adriamycin, d(TTAGGG)4, d(TTAGAG)4, d(TTAGGG)5 and d(TTAGGGT) were analyzed by electrophoretic mobility shift assay. The mobility of d(TTAGGG)3, d(TTAGGG)4 and d(TrAGGG)5 in native polyacrylamide electrophoresis were observed. Methylation protection experiments were performed to investigate the effects of adriamycin on methylation of guanine in d(TTAGGG)4 and d(TTAGAG)4. The traditional telomeric repeats amplification protocol (TRAP) and modified TRAP-G4 assays were, respectively, used to analyze the different characteristcs of adriamycin's inhibiting telomeric extension mediated by telomerase of Tca8113 cells.

RESULTSAt 5.00 microg/mL of adriamycin, conversion of some of linear d(TrAGGG)4 and d(TrAGGG)5to the new, high-mobility bands formed by complex with special second structures were found in the mobility shift assay. Adriamycin at 1.25 microg/mL protected the G in d(TIAGGG)4 from methylating. Adriamycin at 2.50 microg/mL or 1.25 microg/mL partially inhibited the telomeric extension lengthened by telomerase of Tca8113 cells in TRAP assay, but completely did so in TRAP-G4 assay.

CONCLUSIONAdriamycin is able to disturb telomeric extention mediated by telomerase of Tca8113 cells by inducing oligonucleotides that contain telomeric repeats to form intra-molecular G4 structures.

DNA ; Doxorubicin ; G-Quadruplexes ; Guanine ; Nucleic Acid Conformation ; Telomerase ; Telomere

G-quadruplex DNA has become an important target for tumor therapy and anti-tumor development. Modern pharmacology has proved that Macleaya cordata has anti-inflammatory, antibacterial, anti-tumor and other pharmacological effects. Affinity ultrafiltration method can screen active ingredients from compounds rapidly, but G-quadruplex DNA ligands are difficult to dissociate, which is a key step in conventional ultrafiltration method. In this paper, the filtrates after ultrafiltration were determined by HPLC-MS in substitution. The peaks with 20% reduction of MS response from the incubation vs control were considered to be ligand components to G-quadruplex. Two of the peaks with the relative abundance above 30% were identified as sanguinarine(SAN) and chelerine(CHE). Their circular dichroism conformations further proved that SAN and CHE are active ligands of HT4. In addition, another two gradients with high relative abundance were identified as protopine(PRO) and allpcryprotopine(ALL). The binding rate of SAN, CHE, PRO and ALL was calculated according to the HPLC-MS results, and the results showed a consistency with that of the molecular docking method. The proposed method can be used to screen active components from mixture.
Chromatography, High Pressure Liquid ; Chromatography, Liquid ; G-Quadruplexes ; Ligands ; Mass Spectrometry ; Molecular Docking Simulation ; Ultrafiltration

This study was aimed to investigate the growth inhibition effect of diimide G-quadruplex ligand on leukemia cells and to explore its molecular mechanisms. K562 leukemia cell lines were treated with various concentrations of the diimide G-quadruples ligand small molecule (0.1 - 10 micromol/L). Trypan blue exclusion assay was used to evaluate the proliferation inhibition. Cell apoptosis was observed using terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL). Telomerase activity was analyzed by telomere repeat amplification protocol. Gene expression was detected by microarray and confirmed by RT-PCR assay. The results showed that diimide small molecule inhibited the proliferation of K562 cells and induced apoptosis of these cells. After treating with diimide G-quadruplex ligand, telomerase activity of K562 cells was reduced and the transcriptional levels of some important genes were changed significantly. These genes were involved in cell apoptosis, cell signaling pathway and other key functions. In conclusion, the diimide G-quadruplex ligand is a small molecule that inhibits the proliferation and induces apoptosis in leukemia cells, and these functions may be related to telomerase inhibition and regulation of some important gene transcription.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; G-Quadruplexes ; Humans ; K562 Cells ; Leukemia ; genetics ; pathology ; Ligands ; Microarray Analysis ; Telomerase ; metabolism

Telomere assumes intra-molecular G-quadruplex that is a significant drug target for inhibiting telomerase maintenance of telomeres in cancer. Metal cations have been recognized as playing important roles in stabilizing G-quadruplex, but their binding processes to human telomeric G-quadruplex remain uncharacterized. To investigate the detailed binding procedures, molecular dynamics simulations were conducted on the hybrid [3 + 1] form-one human telomeric intra-molecular G-quadruplex. We show here that the binding of a potassium ion to a G-tetrad core is mediated by two alternative pathways. Principal component analysis illustrated the dominant concerted motions of G-quadruplex occurred at the loop domains. MM-PBSA calculations revealed that binding was energetically favorable and driven by the electrostatic interactions. The lower binding site was found more constructive favorable for binding. Our data provide useful information on a potassium-mediated stable structure of human telomeric intra-molecular G-quadruplex, implicating in ion disorder associated conformational changes and targeted drug design.
Binding Sites ; G-Quadruplexes ; Humans ; Molecular Dynamics Simulation ; Movement ; Potassium ; metabolism ; Principal Component Analysis ; Substrate Specificity ; Telomere ; chemistry ; metabolism ; Thermodynamics

This study was aimed to investigate the anti-leukemia activity of Tel03 in vivo. The K562 xenografted leukemia model was established and mice were divided randomly into three groups. Mice of different group were treated with PBS (control), 5 mg/kg Tel03 or 15 mg/kg Tel03 (ip, twice a week) respectively. Tumor volume, body weight and other behavior were observed regularly. Cell apoptosis was detected with TUNEL assay and the expression levels of Bcl-2 and Bax were detected by Western blot. The results indicated that Tel03 exerted anti-leukemia activity in mouse model. Tel03 significantly reduced tumor volume in Tel03-treated group compared with control. In addition, 5 mg/kg Tel03 induced cell apoptosis without exerting apparent toxicity in mice. After Tel03 treatment, the expression of Bcl-2 was inhibited, however, the expression of Bax was up-regulated. It is concluded that G-quadruplex ligand Tel03 can induce cell apoptosis in leukemia mouse model, and this agent may be a potential anticancer drug.
Animals ; Apoptosis ; Female ; G-Quadruplexes ; Humans ; K562 Cells ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; metabolism

The anti-tumor activities and mechanism of Erythrina variegata L. extract were investigated. Firstly, the MTT method was used to evaluate the inhibitory activity of the Erythrina variegata L. extract on proliferation of cancer cell lines. Moreover, in order to determine its anti-tumor effect in vivo, the Lewis lung cancer mice model was established. By comparing the relative tumor proliferation rates, growth curves, inhibition rates of different groups, the anti-tumor effect was evaluated. Furthermore, the anti-tumor mechanism of Erythrina variegata L. extract was studied by using G-quadruplex stability experiment. In the in vitro anti-liver cancer experiment, the Erythrina variegata L. extract has shown obvious anti-tumor effect on various tumor cells. And in the in vivo experiment, it exhibited significant anti-tumor effect. Besides, from the result of G-quadruplex stability experiment, we can see that the quadruplex structure show increasing T(m) values with increasing amounts of Erythrina variegata L. extract.
Animals ; Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Carcinoma, Lewis Lung ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Erythrina ; chemistry ; G-Quadruplexes ; drug effects ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Plants, Medicinal ; chemistry ; Tumor Burden ; drug effects