RESUMEN
WNT1 encodes a multifunctional signaling glycoprotein that is highly expressed in several malignant tumors. Patients with Wnt1-positive cancer are usually related to advanced metastasis. Here, we found that a stretch of G-rich sequences located at the WNT1 promoter region is capable of forming G-quadruplex structures. The addition of G-quadruplex structure stabilizers, BMVC and BMVC4, raises the melting temperature of the oligonucleotide formed by the WNT1 promoter G-rich sequences. Significantly, the expression of WNT1 was repressed by BMVC or BMVC4 in a G-quadruplex-dependent manner, suggesting that they can be used to modulate WNT1 expression. The role of G-quadruplex stabilizers on Wnt1-mediated cancer migration and invasion was further analyzed. The protein levels of ß-catenin, a mediator of the Wnt-mediated signaling pathway, and the downstream targets MMP7 and survivin were down-regulated upon BMVC or BMVC4 treatments. Moreover, the migration and invasion activities of cancer cells were inhibited by BMVC and BMVC4, and the inhibitory effects can be reversed by WNT1-overexpression. Thus the Wnt1 expression and its downstream signaling pathways can be regulated through the G-quadruplex sequences located at its promoter region. These findings provide a novel approach for future drug development to inhibit migration and invasion of cancer cells.
Asunto(s)
Carbazoles/farmacología , Movimiento Celular/efectos de los fármacos , G-Cuádruplex/efectos de los fármacos , Pirazinas/farmacología , Compuestos de Piridinio/farmacología , Proteína Wnt1/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Immunoblotting , Proteínas Inhibidoras de la Apoptosis/genética , Metaloproteinasa 7 de la Matriz/genética , Invasividad Neoplásica , Oligonucleótidos/química , Oligonucleótidos/genética , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Survivin , beta Catenina/metabolismoRESUMEN
Telomerase activity in cancer cells is commonly analyzed by a polymerase chain reaction (PCR)-based assay termed the telomeric repeat amplification protocol (TRAP). However, nonspecific inhibition of Taq polymerase during the PCR step is frequently observed in inhibitor analysis or drug screening. Thus, the removal of excess inhibitors prior to PCR is an essential step for the proper evaluation of telomerase inhibitory effects. Here, a size exclusion spin column was applied to remove small molecular weight inhibitors from the telomerase extension products. The spin column-added protocol, termed sTRAP, provides a more reliable estimation of the inhibitory effects of telomerase activity.
Asunto(s)
Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Telomerasa/antagonistas & inhibidores , Línea Celular Tumoral , Dextranos/química , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Reacción en Cadena de la Polimerasa/métodos , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/farmacología , Polimerasa Taq/antagonistas & inhibidores , Polimerasa Taq/metabolismo , Telomerasa/metabolismoRESUMEN
Four series of compounds containing an anthraquinone-linked moiety and symmetrical or asymmetrical aminoacyl residues in side chains at positions 1,4-, 1,5-, 2,6-, and 2,7- were synthesized and evaluated for their inhibitory effects toward telomerase and hTERT expression. Of these, only compound B11 showed selective inhibition of telomerase activity. Although it is not as competent as several of the anthraquinones we identified previously, nevertheless, the result is consistent with that the general structure moiety at the 1,5-position of diaminoanthraquinone-linked compound is important for the telomerase inhibitory activity. Interestingly, compounds A6, A8, C8, and D8 exhibited selective repressing activities toward hTERT expression and showed less effect toward proliferation of the treated cancer cells. Although it is not apparent which structure moiety is responsible for the telomerase repression effects of these compounds, they could be further developed as potential anti-tumor agents.
Asunto(s)
Antraquinonas/síntesis química , Antineoplásicos/síntesis química , Proliferación Celular/efectos de los fármacos , Telomerasa/antagonistas & inhibidores , Acilación , Aminación , Antraquinonas/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Humanos , Relación Estructura-ActividadRESUMEN
Carbazole derivatives that stabilized G-quadruplex DNA structure formed by human telomeric sequence have been designed and synthesized. Among them, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) showed an increase in G-quadruplex melting temperature by 13 degrees C and has a potent inhibitory effect on telomerase activity. Treatment of H1299 cancer cells with 0.5 mumol/L BMVC did not cause acute toxicity and affect DNA replication; however, the BMVC-treated cells ceased to divide after a lag period. Hallmarks of senescence, including morphologic changes, detection of senescence-associated beta-galactosidase activity, and decreased bromodeoxyuridine incorporation, were detected in BMVC-treated cancer cells. The BMVC-induced senescence phenotype is accompanied by progressive telomere shortening and detection of the DNA damage foci, indicating that BMVC caused telomere uncapping after long-term treatments. Unlike other telomerase inhibitors, the BMVC-treated cancer cells showed a fast telomere shortening rate and a lag period of growth before entering senescence. Interestingly, BMVC also suppressed the tumor-related properties of cancer cells, including cell migration, colony-forming ability, and anchorage-independent growth, indicating that the cellular effects of BMVC were not limited to telomeres. Consistent with the observations from cellular experiments, the tumorigenic potential of cancer cells was also reduced in mouse xenografts after BMVC treatments. Thus, BMVC repressed tumor progression through both telomere-dependent and telomere-independent pathways.
Asunto(s)
Antineoplásicos/farmacología , Carbazoles/farmacología , Senescencia Celular/efectos de los fármacos , G-Cuádruplex/efectos de los fármacos , Compuestos de Piridinio/farmacología , Telomerasa/antagonistas & inhibidores , Telómero/efectos de los fármacos , Animales , Antineoplásicos/química , Carbazoles/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , ADN/química , Ratones , Ratones Endogámicos BALB C , Neoplasias/patología , Compuestos de Piridinio/química , Telómero/químicaRESUMEN
A series of 1,2-heteroannelated anthraquinones and anthra[1,2-d]imidazole-6,11-dione tetracyclic analogues with different side chain were prepared using an various synthetic route via acylation, cyclization, condensation, and intramolecular heterocyclization. Tetracyclic system containing alkyl and aryl, aromatic and heterocyclic, linear and cyclic, polar and apolar, and basic and acids residues were incorporated. They were evaluated for their effects on telomerase activity, hTERT expression, cell proliferations, and in vitro cytotoxicity against NCI's 60 cell line human tumor screen. Compounds 4, 11, 12, 14, 15, 16, 17, 19, 20, 23, 25, and 26 were selected by the NCI for one dose screening program and further studies on 4, 23 and 25 where the curves cross these lines represent the interpolated values to cause 50% growth inhibition (GI(50)), total growth inhibition (TGI) and 50% cell killing (LC(50)), respectively. Further studies did not reveal any compound that showed potent and significant on telomerase inhibitory activity and hTERT repressing ability. Comparative testing of these compounds in the NCI's screen revealed varying levels of potency and differential cytotoxicity, apparently related to the unsaturation levels in and substitution patterns on the core ring system. It appeared that addition of a fourth planar aromatic system to a tricyclic chromophore might enhances potent cytotoxic agents, at a level equivalent to a second side chain in one of the tricyclic series. Although the exact mechanism of how this pharmacophore contributes to its activity is still unclear, however, the group in the extended arm of the tetracyclic system might contribute to proper binding to the residues within the grove of G-quadruplex structure.
Asunto(s)
Antraquinonas/síntesis química , Antineoplásicos/síntesis química , Imidazoles/síntesis química , Inhibidores de la Transcriptasa Inversa/síntesis química , Telomerasa/antagonistas & inhibidores , Tiadiazoles/síntesis química , Antraquinonas/química , Antraquinonas/toxicidad , Antineoplásicos/química , Antineoplásicos/toxicidad , Línea Celular Tumoral , Proliferación Celular , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Imidazoles/química , Imidazoles/toxicidad , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/toxicidad , Relación Estructura-Actividad , Telomerasa/metabolismo , Tiadiazoles/química , Tiadiazoles/toxicidadRESUMEN
We developed the synthesis of a series of thiophene-fused tetracyclic analogues of the antitumor drug ametantrone. The reactions included nucleophilic substitution of methoxy groups in 4,11-dimethoxyanthra[2,3-b]thiophene-5,10-diones with ethylenediamines, producing the derivatives of 4,11-diaminoanthra[2,3-b]thiophene-5,10-dione in good yields. Several compounds showed marked antiproliferative potency against doxorubicin-selected, P-glycoprotein-expressing tumor cells and p53(-/-) cells. The cytotoxicity of some novel compounds for P-glycoprotein-positive cells is highly dependent on N-substituent at the terminal amino group of ethylenediamine moiety. The cytotoxic potency of selected compounds correlated with their ability to attenuate the functions of topoisomerase I and telomerase, strongly suggesting that these enzymes are the major targets of antitumor activity of anthra[2,3-b]thiophene-5,10-dione derivatives.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/toxicidad , Mitoxantrona/análogos & derivados , Antineoplásicos/química , Línea Celular Tumoral , ADN-Topoisomerasas de Tipo I/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Concentración 50 Inhibidora , Mitoxantrona/síntesis química , Mitoxantrona/química , Mitoxantrona/toxicidad , Inhibidores de Topoisomerasa IRESUMEN
Telomerase is important in tumor initiation and cellular immortalization. Given the striking correlations between telomerase activity and proliferation capacity in tumor cells, telomerase had been considered as a potentially important molecular target in cancer therapeutics. A series of 2,7-diamidoanthraquinone were designed and synthesized. They were evaluated for their effects on telomerase activity, hTERT expression, cell proliferations, and cytotoxicity. In the series, compounds (6, 10, 13, 16, 18, 19, 20-22, and 24) showed potent telomerase inhibitory activity, while compounds 19, 21, and 22 activated hTERT expression in normal human fibroblasts. The results indicated that 2,7-diamidoanthraquinones represent an important class of compounds for telomerase-related drug developments. Compounds 8, 16, 18, 26, and 32 were also selected by the NCI for Screening Program and demonstrated high anti-proliferative activity against 60 human cancer cell lines. Structure-activity relationships (SAR) study revealed that the test compounds with side chains two carbon spacer between amido and amine are important structural moiety for telomerase inhibition. Although the exact mechanism of how this amine group contributes to its activity is still unclear, however, the amine group in the extended arm of the bis-substituted anthraquinone might contribute to proper binding to the residues within the grove of G-quadruplex structure. Our results indicated that the 2,7-disubstituted amido-anthraquinones are potent telomerase inhibitors that have the potential to be further developed into novel anticancer chemotherapeutic agents.
Asunto(s)
Antraquinonas/síntesis química , Antraquinonas/farmacología , Antineoplásicos/síntesis química , Telomerasa/antagonistas & inhibidores , Antraquinonas/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ensayos de Selección de Medicamentos Antitumorales , Fibroblastos/efectos de los fármacos , Humanos , Relación Estructura-Actividad , Telomerasa/efectos de los fármacosRESUMEN
A series of anthra[1,2-d]imidazole-6,11-dione derivatives were synthesized and evaluated for telomerase inhibition, hTERT expression and suppression of cancer cell growth in vitro. All of the compounds tested, except for compounds 4, 7, 16, 24, 27 and 28 were selected by the NCI screening system. Among them, compounds 16, 39, and 40 repressed hTERT expression without greatly affecting cell growth, suggesting for the selectivity toward hTERT expression. Taken together, our findings indicated that the analysis of cytotoxicity and telomerase inhibition might provide information applicable for further developing potential telomerase and polypharmacological targeting strategy.
Asunto(s)
Antineoplásicos/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Telomerasa/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Relación Estructura-Actividad , Telomerasa/genética , Telomerasa/metabolismo , Células Tumorales CultivadasRESUMEN
By using fragment-based design strategies, a series of 2-thio-substituted anthra[1,2-d]imidazole-6,11-diones were synthesized and evaluated for hTERT repressing activities, cell proliferations, and NCI 60-cell panel assay. Compounds 2, 3, 4, 11, 15 and 35 were selected by the NCI and 3, 4, 11 and 15 represent the GI50, TGI and LC50, respectively. Among them, all were moderate selectivity toward leukemia cancer except for 4 exhibited distinctive selectivity of CNS and renal cancer with 7.403 and 6.475. The overall of test compounds exhibited different cytostatic and cytotoxic activities for further developing potential application as anticancer drugs.
Asunto(s)
Antraquinonas/farmacología , Antineoplásicos/farmacología , Bencimidazoles/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Telomerasa/antagonistas & inhibidores , Antraquinonas/síntesis química , Antraquinonas/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Bencimidazoles/síntesis química , Bencimidazoles/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Compuestos Heterocíclicos de 4 o más Anillos/química , Humanos , Estructura Molecular , Relación Estructura-Actividad , Telomerasa/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Telomerase is the enzyme responsible for extending G-strand telomeric DNA and represents a promising target for treatment of neoplasia. Inhibition of telomerase can be achieved by stabilization of G-quadruplex DNA structures. Here, we characterize the cellular effects of a novel G-quadruplex stabilizing compound, 3,6-bis(4-methyl-2-vinylpyrazinium iodine) carbazole (BMVC4). EXPERIMENTAL APPROACH: The cellular effects of BMVC4 were characterized in both telomerase-positive and alternative lengthening of telomeres (ALT) cancer cells. The molecular mechanism of how BMVC4 induced senescence is also addressed. KEY RESULTS: BMVC4-treated cancer cells showed typical senescence phenotypes. BMVC4 induced senescence in both ALT and telomerase-overexpressing cells, suggesting that telomere shortening through telomerase inhibition might not be the cause for senescence. A large fraction of DNA damage foci was not localized to telomeres in BMVC4-treated cells and BMVC4 suppressed c-myc expression through stabilizing the G-quadruplex structure located at its promoter. These results indicated that the cellular targets of BMVC4 were not limited to telomeres. Further analyses showed that BMVC4 induced DNA breaks and activation of ataxia telangiectasia-mutated mediated DNA damage response pathway. CONCLUSIONS AND IMPLICATIONS: BMVC4, a G-quadruplex stabilizer, induced senescence by activation of pathways of response to DNA damage that was independent of its telomerase inhibitory activity. Thus, BMVC4 has the potential to be developed as a chemotherapeutic agent against both telomerase positive and ALT cancer cells.
Asunto(s)
Antineoplásicos/farmacología , Carbazoles/farmacología , Senescencia Celular/efectos de los fármacos , G-Cuádruplex , Pirazinas/farmacología , Telomerasa/antagonistas & inhibidores , Carbazoles/química , Línea Celular Tumoral , Daño del ADN , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes myc/genética , Genes myc/fisiología , Humanos , Estructura Molecular , Mutagénesis Sitio-Dirigida , Pirazinas/química , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
A series of diversely asymmetrical mono- or disubstituted 1,2-diamidoanthraquinone derivatives were synthesized and evaluated for drug-induced cytotoxicity by SRB assay, telomerase inhibitory activity by TRAP assay, and hTERT expression by SEAP assay. Interestingly, compounds 4, 11, 21, 32 and 36 exhibited selective potent antiproliferative activities by NCI with IC(50) values in the micromolar range. Of these, only compound 8 showed an IC(50) value of 0.95 µM against PC-3 cell lines (human prostate cancer) by SRB assay. All the synthesized compounds exhibited a poor or modest telomerase inhibitory activity by TRAP assay suggesting another mode of action for these compounds. Compound 11 showed broad inhibition against different types of cancer cell lines in the micromolar and submicromolar range.
Asunto(s)
Antraquinonas/química , Antraquinonas/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Telomerasa/antagonistas & inhibidores , Antraquinonas/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Humanos , Concentración 50 Inhibidora , Telomerasa/metabolismoRESUMEN
A series of symmetrical disubstituded 1,8-diamidoanthraquinones were synthesized and evaluated for anti-proliferation, telomerase inhibitory by TRAP assay, and hTERT expression by SEAP assay. All of the compounds tested, except for compounds 3a and 3s were selected by the NCI screening system. In addition, compounds 4e and 4k exhibited inhibitory effects on telomerase activity. Taken together, our findings indicated that the analysis of cytotoxicity and telomerase inhibition might provide information applicable for further developing potential telomerase targeting strategy.
Asunto(s)
Antraquinonas/química , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Neoplasias/tratamiento farmacológico , Telomerasa/antagonistas & inhibidores , Fosfatasa Alcalina/metabolismo , Apoptosis/efectos de los fármacos , Humanos , Telomerasa/metabolismo , Células Tumorales CultivadasRESUMEN
Telomerase is the enzymatic activity that maintains the ends of eukaryotic chromosomes. Telomerase activity is detected in most tumor cells whereas it is low or undetectable in most normal somatic cells. Expression of the telomerase catalytic component, the human telomerase reverse transcriptase (hTERT), is believed to be controlled primarily at the level of transcription. Because of this selective expression property of telomerase, it has been touted as a specific target for antitumor chemotherapeutics. However, a concern for the applicability of telomerase inhibitors is that they require a long lag time for telomeres to be shortened to critical length before cancer cells stop proliferating. Here we investigate telomerase inhibitory, cytotoxicity and the hTERT repressing effects on a number of synthesized 2,6-diamidoanthraquinones and 1,5-diamidoanthraquinones as compared to their disubstituted homologues. We found that several of the 1,5-diamidoanthraquinones and 2,6-diamidoanthraquinones inhibited telomerase activity effectively with IC50 at the sub-micro to micro molar range and caused acute cytotoxicity to cancer cells with EC50 similar or better than that of mitoxantrone. Particularly, 2,6-diamidoanthraquinone with 2-ethylaminoacetamido side chains 33, even though not affecting cell proliferation, showed to be endowed with a strong telomerase effect, probably related to a marked stabilization of the G-quadruplex-binding structure. The results suggested that these compounds caused multiple effects to cancer cells. More significantly, they overcome the long lag period problem of classical telomerase inhibitors that they are also potent cytotoxic agents. These results greatly expand the potential of tricyclic anthraquinone pharmacophore in preventive and/or curative therapy.