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1.
Oncotarget ; 6(17): 15265-82, 2015 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-25948775

RESUMO

5-azacytidine and 5-aza-2'-deoxycytidine are clinically used to treat patients with blood neoplasia. Their antileukemic property is mediated by the trapping and the subsequent degradation of a family of proteins, the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) leading to DNA demethylation, tumor suppressor gene re-expression and DNA damage. Here we studied the respective role of each DNMT in the human leukemia KG1 cell line using a RNA interference approach. In addition we addressed the role of DNA damage formation in DNA demethylation by 5-aza-2'-deoxycytidine. Our data show that DNMT1 is the main DNMT involved in DNA methylation maintenance in KG1 cells and in mediating DNA damage formation upon exposure to 5-aza-2'-deoxycytidine. Moreover, KG1 cells express the DNMT1 protein at a level above the one required to ensure DNA methylation maintenance, and we identified a threshold for DNMT1 depletion that needs to be exceeded to achieve DNA demethylation. Most interestingly, by combining DNMT1 siRNA and treatment with low dose of 5-aza-2'-deoxycytidine, it is possible to uncouple DNA damage formation from DNA demethylation. This work strongly suggests that a direct pharmacological inhibition of DNMT1, unlike the use of 5-aza-2'-deoxycytidine, should lead to tumor suppressor gene hypomethylation and re-expression without inducing major DNA damage in leukemia.


Assuntos
Azacitidina/análogos & derivados , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Leucemia/tratamento farmacológico , Azacitidina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferase 1 , Dano ao DNA/genética , Metilação de DNA/efeitos dos fármacos , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/genética , Decitabina , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Histonas/metabolismo , Humanos , Proteínas Nucleares/genética , Fosforilação , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Interferente Pequeno , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/genética , DNA Metiltransferase 3B
2.
ChemMedChem ; 9(3): 590-601, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24678024

RESUMO

Quinoline derivative SGI-1027 (N-(4-(2-amino-6-methylpyrimidin-4-ylamino)phenyl)-4-(quinolin-4-ylamino)benzamide) was first described in 2009 as a potent inhibitor of DNA methyltransferase (DNMT) 1, 3A and 3B. Based on molecular modeling studies, performed using the crystal structure of Haemophilus haemolyticus cytosine-5 DNA methyltransferase (MHhaI C5 DNMT), which suggested that the quinoline and the aminopyridimine moieties of SGI-1027 are important for interaction with the substrates and protein, we designed and synthesized 25 derivatives. Among them, four compounds­namely the derivatives 12, 16, 31 and 32­exhibited activities comparable to that of the parent compound. Further evaluation revealed that these compounds were more potent against human DNMT3A than against human DNMT1 and induced the re-expression of a reporter gene, controlled by a methylated cytomegalovirus (CMV) promoter, in leukemia KG-1 cells. These compounds possessed cytotoxicity against leukemia KG-1 cells in the micromolar range, comparable with the cytotoxicity of the reference compound, SGI-1027. Structure­activity relationships were elucidated from the results. First, the presence of a methylene or carbonyl group to conjugate the quinoline moiety decreased the activity. Second, the size and nature of the aromatic or heterocycle subsitutents effects inhibition activity: tricyclic moieties, such as acridine, were found to decrease activity, while bicyclic substituents, such as quinoline, were well tolerated. The best combination was found to be a bicyclic substituent on one side of the compound, and a one-ring moiety on the other side. Finally, the orientation of the central amide bond was found to have little effect on the biological activity. This study provides new insights in to the structure-activity relationships of SGI-1027 and its derivative.


Assuntos
Aminoquinolinas/síntese química , Aminoquinolinas/farmacologia , Metilação de DNA/efeitos dos fármacos , Desenho de Fármacos , Pirimidinas/síntese química , Pirimidinas/farmacologia , Quinolinas/química , Aminoquinolinas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Pirimidinas/química , Relação Estrutura-Atividade
3.
Biochimie ; 94(11): 2280-96, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22967704

RESUMO

This review presents the different human DNA methyltransferases (DNMTs), their biological roles, their mechanisms of action and their role in cancer. The description of assays for detecting DNMT inhibitors (DNMTi) follows. The different known DNMTi are reported along with their advantages, drawbacks and clinical trials. A discussion on the features of the future DNMT inhibitors will conclude this review.


Assuntos
Metilação de DNA/efeitos dos fármacos , Neoplasias/genética , Animais , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia
4.
Bioorg Med Chem ; 20(2): 819-31, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22206869

RESUMO

The interesting pharmacological properties of neoboutomellerones 1 and 2 were the basis for the assembly of a small library of analogues consisting of natural products isolated from the plant Neoboutonia melleri and of semisynthetic derivatives. As the two enone systems (C23-C24a and C1-C3) and the two hydroxyls groups (C22 and C26) of neoboutomellerones are required for activity, modifications were focused on these functional groups. Biological evaluation by using a cellular assay for proteasome activity provided clues regarding the mechanism of action of these natural products and synthetic derivatives. Certain neoboutomellerone derivatives inhibited the proliferation of human WM-266-4 melanoma tumor cells at submicromolar concentration and warrant evaluation as anticancer agents.


Assuntos
Antineoplásicos/síntese química , Produtos Biológicos/química , Inibidores de Proteassoma , Triterpenos/síntese química , Ubiquitina/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Euphorbiaceae/química , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Triterpenos/química , Triterpenos/toxicidade , Ubiquitina/metabolismo
5.
Biochem Pharmacol ; 82(12): 1843-52, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21924246

RESUMO

The polyamines transport system (PTS) is usually enhanced in cancer cells and can be exploited to deliver anticancer drugs. The spermine-conjugated epipodophyllotoxin derivative F14512 is a topoisomerase II poison that exploits the PTS to target preferentially tumor cells. F14512 has been characterized as a potent anticancer drug candidate and is currently in phase 1 clinical trials. Here we have analyzed the mechanisms of cell death induced by F14512, compared to the parent drug etoposide lacking the polyamine tail. F14512 proved to be >30-fold more cytotoxic than etoposide against A549 non-small cell lung cancer cells and triggers less but unrecoverable DNA damages. The cytotoxic action of F14512 is extremely rapid (within 3 h) and does not lead to a marked accumulation in the S-phase of the cell cycle, unlike etoposide. Interestingly, A549 cells treated with F14512 were less prone to undergo apoptosis (neither caspases-dependent nor caspases-independent pathways) or autophagy but preferentially entered into senescence. Drug-induced senescence was characterized qualitatively and quantitatively by an increased ß-galactosidase activity, both by cytochemical staining and by flow cytometry. A morphological analysis by electron microscopy revealed the presence of numerous multi-lamellar and vesicular bodies and large electron-lucent (methuosis-like) vacuoles in F14512-treated cell samples. The mechanism of drug-induced cell death is thus distinct for F14512 compared to etoposide, and this difference may account for their distinct pharmacological profiles and the markedly superior activity of F14512 in vivo. This study suggests that senescence markers should be considered as potential pharmacodynamic biomarkers of F14512 antitumor activity.


Assuntos
Antineoplásicos/farmacologia , DNA Topoisomerases Tipo II/metabolismo , Podofilotoxina/análogos & derivados , Poliaminas/metabolismo , Inibidores da Topoisomerase II/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Ciclo Celular , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Etoposídeo/química , Etoposídeo/farmacologia , Humanos , Estrutura Molecular , Podofilotoxina/química , Podofilotoxina/farmacologia
6.
PLoS One ; 6(8): e23597, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21853156

RESUMO

F14512 is a novel anti-tumor molecule based on an epipodophyllotoxin core coupled to a cancer-cell vectoring spermine moiety. This polyamine linkage is assumed to ensure the preferential uptake of F14512 by cancer cells, strong interaction with DNA and potent inhibition of topoisomerase II (Topo II). The antitumor activity of F14512 in human tumor models is significantly higher than that of other epipodophyllotoxins in spite of a lower induction of DNA breakage. Hence, the demonstrated superiority of F14512 over other Topo II poisons might not result solely from its preferential uptake by cancer cells, but could also be due to unique effects on Topo II interactions with DNA. To further dissect the mechanism of action of F14512, we used Drosophila melanogaster mutants whose genetic background leads to an easily scored phenotype that is sensitive to changes in Topo II activity and/or localization. F14512 has antiproliferative properties in Drosophila cells and stabilizes ternary Topo II/DNA cleavable complexes at unique sites located in moderately repeated sequences, suggesting that the drug specifically targets a select and limited subset of genomic sequences. Feeding F14512 to developing mutant Drosophila larvae led to the recovery of flies expressing a striking phenotype, "Eye wide shut," where one eye is replaced by a first thoracic segment. Other recovered F14512-induced gain- and loss-of-function phenotypes similarly correspond to precise genetic dysfunctions. These complex in vivo results obtained in a whole developing organism can be reconciled with known genetic anomalies and constitute a remarkable instance of specific alterations of gene expression by ingestion of a drug. "Drosophila-based anticancer pharmacology" hence reveals unique properties for F14512, demonstrating the usefulness of an assay system that provides a low-cost, rapid and effective complement to mammalian models and permits the elucidation of fundamental mechanisms of action of candidate drugs of therapeutic interest in humans.


Assuntos
Antineoplásicos/farmacologia , DNA Topoisomerases Tipo II/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Podofilotoxina/análogos & derivados , Poliaminas/química , Inibidores da Topoisomerase II/farmacologia , Animais , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Efeitos da Posição Cromossômica/efeitos dos fármacos , DNA/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica/efeitos dos fármacos , Genes de Insetos/genética , Histonas/genética , Humanos , Modelos Animais , Fenótipo , Podofilotoxina/química , Podofilotoxina/farmacologia , Sequências Repetitivas de Ácido Nucleico/genética , Supressão Genética/efeitos dos fármacos , Inibidores da Topoisomerase II/química , Cromossomo X/genética
7.
Planta Med ; 77(14): 1605-9, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21425033

RESUMO

Six carvotanacetone derivatives (1- 6), amongst which four new compounds (1- 4), were isolated from the aerial parts of Sphaeranthus ukambensis Vatke & O. Hoffm. The structures of the molecules were elucidated by complementary spectroscopic methods, and their biological properties were investigated using human DLD-1 colon cancer cells engineered to stably express a 4 ubiquitin-luciferase (4 Ub-Luc) reporter protein. Five of the isolated carvotanacetone derivatives (2- 6) were found to inhibit the proliferation of the colon cancer cells and interfere with the ubiquitin-proteasome pathway, with potencies in a micromolar range.


Assuntos
Asteraceae/química , Extratos Vegetais/química , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Terpenos/farmacologia , Ubiquitina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Quênia , Componentes Aéreos da Planta/química , Terpenos/química , Terpenos/isolamento & purificação
8.
Mol Cancer Ther ; 8(10): 2780-90, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19808979

RESUMO

Triptolide, a natural product extracted from the Chinese plant Tripterygium wilfordii, possesses antitumor properties. Despite numerous reports showing the proapoptotic capacity and the inhibition of NF-kappaB-mediated transcription by triptolide, the identity of its cellular target is still unknown. To clarify its mechanism of action, we further investigated the effect of triptolide on RNA synthesis in the human non-small cell lung cancer cell line A549. Triptolide inhibited both total RNA and mRNA de novo synthesis, with the primary action being on the latter pool. We used 44K human pan-genomic DNA microarrays and identified the genes primarily affected by a short treatment with triptolide. Among the modulated genes, up to 98% are down-regulated, encompassing a large array of oncogenes including transcription factors and cell cycle regulators. We next observed that triptolide induced a rapid depletion of RPB1, the RNA polymerase II main subunit that is considered a hallmark of a transcription elongation blockage. However, we also show that triptolide does not directly interact with the RNA polymerase II complex nor does it damage DNA. We thus conclude that triptolide is an original pharmacologic inhibitor of RNA polymerase activity, affecting indirectly the transcription machinery, leading to a rapid depletion of short-lived mRNA, including transcription factors, cell cycle regulators such as CDC25A, and the oncogenes MYC and Src. Overall, the data shed light on the effect of triptolide on transcription, along with its novel potential applications in cancers, including acute myeloid leukemia, which is in part driven by the aforementioned oncogenic factors.


Assuntos
Diterpenos/química , Regulação para Baixo/efeitos dos fármacos , Fenantrenos/química , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase I/antagonistas & inibidores , Transcrição Gênica/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Diterpenos/farmacologia , Compostos de Epóxi/química , Compostos de Epóxi/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Fenantrenos/farmacologia , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Fatores de Tempo , Proteína Supressora de Tumor p53 , Proteínas Elk-1 do Domínio ets/metabolismo
9.
Bioorg Med Chem Lett ; 19(9): 2474-7, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19332372

RESUMO

The synthesis of a series of conjugated spermine derivatives with benzoxadiazole, phenylxanthene or bodipy fluorophores is described. These fluorescent probes were used to identify the activity of the polyamine transport system (PTS). N(1)-Methylspermine NBD conjugate 5 proved to have the optimal fluorescence characteristics and was used to show a selectivity for PTS-proficient CHO versus PTS-deficient CHO-MG cells. It can therefore be used as a tool for the selection of cells sensitive to cytotoxic compounds vectored through the PTS.


Assuntos
Compostos de Boro/química , Química Farmacêutica/métodos , Corantes Fluorescentes/farmacologia , Oxidiazóis/síntese química , Rodaminas/química , Espermina/síntese química , Animais , Transporte Biológico , Células CHO , Cricetinae , Cricetulus , Desenho de Fármacos , Corantes Fluorescentes/química , Humanos , Oxidiazóis/farmacologia , Poliaminas/química , Espermina/análogos & derivados , Espermina/química , Espermina/farmacologia
10.
Anticancer Drugs ; 20(5): 364-72, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19322071

RESUMO

The anaplastic lymphoma kinase (ALK) is a validated target for the therapy of different malignancies. Aberrant expression of constitutively active ALK chimeric proteins has been implicated in the pathogenesis of anaplastic large-cell lymphoma (ALCL) and has been detected in other cancers such as inflammatory myofibroblastic tumors, diffuse large B-cell lymphomas, certain non-small-cell lung cancers, rhabdomyosarcomas, neuroblastomas and glioblastomas. In the course of a screening program aimed at identifying kinase inhibitors with novel scaffolds, the two pyridoisoquinoline derivatives F91873 and F91874, were identified as multikinase inhibitors with activity against ALK in a biochemical screen. F91873 and F91874 also inhibited nucleophosmin-ALK and signal transducer and activator of transcription 3 phosphorylation in the ALCL cell line COST with the same potency. Both F91873 and F91874 behaved as ATP noncompetitive inhibitors and inhibited cell proliferation of the ALK(+) ALCL cell lines COST, PIO, and Karpas299 ALCL. This growth inhibition effect was associated with a G1-phase cell cycle arrest. Furthermore, administration of F91874 to severe combined immunodeficient mice bearing COST tumor xenografts resulted in a significant antitumor efficacy at 15 mg/kg/day, illustrating the potential utility of such compounds in the treatment of ALK-related pathologies.


Assuntos
Antineoplásicos/uso terapêutico , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinolizinas/uso terapêutico , Tiazóis/uso terapêutico , Quinase do Linfoma Anaplásico , Animais , Antineoplásicos/síntese química , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/enzimologia , Feminino , Fase G1/efeitos dos fármacos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Linfoma Anaplásico de Células Grandes/enzimologia , Linfoma Anaplásico de Células Grandes/patologia , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Inibidores de Proteínas Quinases/síntese química , Estrutura Terciária de Proteína , Quinolizinas/síntese química , Receptores Proteína Tirosina Quinases , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Tiazóis/síntese química , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cancer Res ; 68(23): 9845-53, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19047165

RESUMO

The polyamine transport system (PTS) is an energy-dependent machinery frequently overactivated in cancer cells with a high demand for polyamines. We have exploited the PTS to selectively deliver a polyamine-containing drug to cancer cells. F14512 combines an epipodophyllotoxin core-targeting topoisomerase II with a spermine moiety introduced as a cell delivery vector. The polyamine tail supports three complementary functions: (a) facilitate formulation of a water-soluble compound, (b) increase DNA binding to reinforce topoisomerase II inhibition, and (c) facilitate selective uptake by tumor cells via the PTS. F14512 is 73-fold more cytotoxic to Chinese hamster ovary cells compared with CHO-MG cells with a reduced PTS activity. A decreased sensitivity of L1210 leukemia cells to F14512 was observed in the presence of putrescine, spermidine, and spermine. In parallel, the spermine moiety considerably enhances the drug-DNA interaction, leading to a reinforced inhibition of topoisomerase II. The spermine tail of F14512 serves as a cell delivery vehicle as well as a DNA anchor, and this property translates at the cellular level into a distinct pharmacologic profile. Twenty-nine human solid or hematologic cell lines were used to characterize the high cytotoxic potential of F14512 (median IC50 of 0.18 micromol/L). Finally, the potent antitumor activity of F14512 in vivo was evidenced with a MX1 human breast tumor xenograft model, with partial and complete tumor regressions. This work supports the clinical development of F14512 as a novel targeted cytotoxic drug and sheds light on the concept of selective delivery of drugs to tumor cells expressing the PTS.


Assuntos
Poliaminas Biogênicas/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Podofilotoxina/análogos & derivados , Inibidores da Topoisomerase II , Animais , Ligação Competitiva , Poliaminas Biogênicas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Dano ao DNA , DNA Topoisomerases Tipo II/biossíntese , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , DNA de Neoplasias/metabolismo , Sistemas de Liberação de Medicamentos , Etoposídeo/farmacologia , Feminino , Humanos , Leucemia L1210/tratamento farmacológico , Leucemia L1210/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Neoplasias/enzimologia , Neoplasias/genética , Podofilotoxina/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Biochem Pharmacol ; 76(4): 453-62, 2008 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-18577376

RESUMO

The ubiquitin-proteasome pathway plays a critical role in the degradation of proteins involved in tumor growth and has therefore become a target for cancer therapy. In order to discover novel inhibitors of this pathway, a cellular assay reporter of proteasome activity was established. Human DLD-1 colon cancer cells were engineered to express a 4 ubiquitin-luciferase (DLD-1 4Ub-Luc) reporter protein, rapidly degraded via the ubiquitin-proteasome pathway and designed DLD-1 4Ub-Luc cells. Following treatment with reference proteasome inhibitors, the 4Ub-Luc protein accumulated in DLD-1 4Ub-Luc cells and a 80-fold increase in luciferase-produced bioluminescence signal was measured, as compared to untreated cells. The screening of over 30,000 compounds using this DLD-1 4Ub-Luc assay led to the identification of physalin B as a novel inhibitor of the ubiquitin-proteasome pathway. Indeed, physalin B induced an increase in bioluminescence from DLD-1 4Ub-Luc cells, at concentrations also producing an accumulation of ubiquitinated proteins and inhibiting TNFalpha-induced NF-kappaB activation. Physalin B did not inhibit catalytic activities of purified proteasome and interfered with cellular proteasomal catalytic activities at 4- to 8-fold higher concentrations than that required to induce significant increase in bioluminescence and accumulation of ubiquitinated proteins in DLD-1 4Ub-Luc cells. Furthermore, physalin B proved to be cytotoxic, triggered apoptosis in DLD-1 4Ub-Luc cells and induced the proapoptotic protein NOXA, characteristic of the proteasome signaling pathway. Therefore, the use of the DLD-1 4Ub-Luc assay allowed the identification of a novel inhibitor of the ubiquitin-proteasome pathway that might interfere with proteasome functions in a different way from reference proteasome inhibitors.


Assuntos
Apoptose/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Secoesteroides/farmacologia , Ubiquitina/metabolismo , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Extratos Vegetais/química , Inibidores de Proteases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Transdução de Sinais
13.
Bioorg Med Chem Lett ; 18(3): 1212-6, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18083028

RESUMO

Novel derivatives of the marine alkaloid bengacarboline have been synthesized. The seco derivatives 11 and 12 were evaluated for topoisomerase inhibition, DNA damages, cytotoxicity and cell cycle perturbation. The two synthetic analogs are more potent cytotoxic agents than bengacarboline and they both induce an accumulation of cells in the S phase of DNA synthesis. They do not function as topoisomerase inhibitors but trigger DNA damages in cells.


Assuntos
Alcaloides/síntese química , Alcaloides/farmacologia , Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Carbolinas/síntese química , Carbolinas/farmacologia , Inibidores da Topoisomerase II , Alcaloides/química , Animais , Antineoplásicos Fitogênicos/química , Apoptose/fisiologia , Carbolinas/química , Ciclo Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Biologia Marinha , Estrutura Molecular , Urocordados/química
14.
J Biol Chem ; 280(1): 448-57, 2005 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-15498776

RESUMO

Human immunodeficiency virus, type 1 (HIV-1) transcription is regulated by a virus-encoded protein, Tat, which forms a complex with a host cellular factor, positive transcription elongation factor b (P-TEFb). When this complex binds to TAR RNA synthesized from the HIV-1 long terminal repeat promoter element, transcription is trans-activated. In this study we showed that, in host cells, HIV-1 transcription is negatively regulated by competition of poly(ADP-ribose) polymerase-1 (PARP-1) with Tat.P-TEFb for binding to TAR RNA. PARP-1, which has a high affinity for TAR RNA (K(D) = 1.35 x 10(-10) M), binds to the loop region of TAR RNA and displaces Tat or Tat.P-TEFb from the RNA. In vitro transcription assays showed that this displacement leads to suppression of Tat-mediated trans-activation of transcription. Furthermore in vivo expression of luciferase or destabilized enhanced green fluorescent protein genes under the control of the HIV-1 long terminal repeat promoter was suppressed by PARP-1. Thus, these results suggest that PARP-1 acts as a negative regulator of HIV-1 transcription through competitive binding with Tat or the Tat.P-TEFb complex to TAR RNA.


Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Poli(ADP-Ribose) Polimerases/genética , Fator B de Elongação Transcricional Positiva/genética , Proteínas de Ligação a RNA/genética , Replicação Viral , Ligação Competitiva , Linhagem Celular , Infecções por HIV/genética , Infecções por HIV/metabolismo , Humanos , Substâncias Macromoleculares/metabolismo , Proteínas Nucleares , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , Ligação Proteica , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica
15.
J Biol Chem ; 278(37): 35279-85, 2003 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-12832398

RESUMO

Double-strand DNA breaks are the most lethal type of DNA damage induced by ionizing radiations. Previously, we reported that double-strand DNA breaks can be enzymatically produced from two DNA damages located on opposite DNA strands 18 or 30 base pairs apart in a cell-free double-strand DNA break formation assay (Vispé, S., and Satoh, M. S. (2000) J. Biol. Chem. 275, 27386-27392). In the assay that we developed, these two DNA damages are converted into single-strand interruptions by enzymes involved in base excision repair. We showed that these single-strand interruptions are converted into double-strand DNA breaks; however, it was not due to spontaneous denaturation of DNA. Thus, we proposed a model in which DNA polymerase delta/epsilon, by producing repair patches at single-strand interruptions, collide, resulting in double-strand DNA break formation. We tested the model and investigated whether other enzymes/factors are involved in double-strand DNA break formation. Here we report that, instead of DNA polymerase delta/epsilon, flap endonuclease-1 (FEN-1), an enzyme involved in base excision repair, is responsible for the formation of double-strand DNA break in the assay. Furthermore, by transfecting a flap endonuclease-1 expression construct into cells, thus altering their flap endonuclease-1 content, we found an increased number of double-strand DNA breaks after gamma-ray irradiation of these cells. These results suggest that flap endonuclease-1 acts as a double-strand DNA break formation factor. Because FEN-1 is an essential enzyme that plays its roles in DNA repair and DNA replication, DSBs may be produced in cells as by-products of the activity of FEN-1.


Assuntos
Dano ao DNA , Reparo do DNA/fisiologia , DNA/efeitos da radiação , Endodesoxirribonucleases/metabolismo , Animais , Sequência de Bases , Linhagem Celular , DNA/química , Endonucleases Flap , Raios gama , Células HeLa , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Proteínas Recombinantes/metabolismo
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