RESUMO
Although many advances have been achieved to treat aggressive tumours, cancer remains a leading cause of death and a public health problem worldwide. Among the main approaches for the discovery of new bioactive agents, the prospect of microbial secondary metabolites represents an effective source for the development of drug leads. In this study, we investigated the actinobacterial diversity associated with an endemic Antarctic species, Deschampsia antarctica, by integrated culture-dependent and culture-independent methods and acknowledged this niche as a reservoir of bioactive strains for the production of antitumour compounds. The 16S rRNA-based analysis showed the predominance of the Actinomycetales order, a well-known group of bioactive metabolite producers belonging to the Actinobacteria phylum. Cultivation techniques were applied, and 72 psychrotolerant Actinobacteria strains belonging to the genera Actinoplanes, Arthrobacter, Kribbella, Mycobacterium, Nocardia, Pilimelia, Pseudarthrobacter, Rhodococcus, Streptacidiphilus, Streptomyces and Tsukamurella were identified. The secondary metabolites were screened, and 17 isolates were identified as promising antitumour compound producers. However, the bio-guided assay showed a pronounced antiproliferative activity for the crude extracts of Streptomyces sp. CMAA 1527 and Streptomyces sp. CMAA 1653. The TGI and LC50 values revealed the potential of these natural products to control the proliferation of breast (MCF-7), glioblastoma (U251), lung/non-small (NCI-H460) and kidney (786-0) human cancer cell lines. Cinerubin B and actinomycin V were the predominant compounds identified in Streptomyces sp. CMAA 1527 and Streptomyces sp. CMAA 1653, respectively. Our results suggest that the rhizosphere of D. antarctica represents a prominent reservoir of bioactive actinobacteria strains and reveals it as an important environment for potential antitumour agents.
Assuntos
Actinobacteria , Técnicas de Cultura/métodos , Descoberta de Drogas , Neoplasias/patologia , Actinobacteria/metabolismo , Actinomycetales/metabolismo , Regiões Antárticas , Antraciclinas/isolamento & purificação , Antraciclinas/metabolismo , Antraciclinas/farmacologia , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Fatores Biológicos/biossíntese , Fatores Biológicos/isolamento & purificação , Fatores Biológicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dactinomicina/biossíntese , Dactinomicina/isolamento & purificação , Dactinomicina/farmacologia , Humanos , Streptomyces/metabolismoRESUMO
OBJECTIVE: To evaluate the impact of periodic shortage of actinomycin-d (Act-d) in the treatment of Brazilian patients with low-risk gestational trophoblastic neoplasia (GTN) after methotrexate and folinic acid rescue (MTX/FA) resistance, treated alternately with carboplatin or etoposide as a second-line regimen. METHODS: Retrospective cohort that included patients with failure of first-line MTX/FA regimen for low-risk GTN treated at Rio de Janeiro Federal University, Universidade Federal de São Paulo and Irmandade da Santa Casa de Misericórdia de Porto Alegre, from January/2010- December/2017. RESULTS: From 356 patients with low-risk GTN treated with MTX/FA, 75 (21.1%) developed resistance, of which 40 (53.3%) received Act-d, 23 (30.7%) carboplatin and 7 (9.3%) etoposide. Although patients treated with single-agent chemotherapy as a second-line regimen had comparable clinical and primary treatment characteristics, those treated with Act-d (80%, pâ¯=â¯0.033) or etoposide (71.4%, pâ¯=â¯0.025) had higher remission rates when compared with carboplatin (47.8%). Only 29% of patients treated with carboplatin received the chemotherapy cycles without delay compared to Act-d (98%, pâ¯<â¯0.001) or etoposide (85%, pâ¯=â¯0.009). Patients treated with carboplatin had significantly more hematological toxicity, notably anemia (30.4%, pâ¯=â¯0.008), lymphopenia (47.7%, pâ¯<â¯0.001) and thrombocytopenia (43.4%, pâ¯<â¯0.001), as well as a higher occurrence of febrile neutropenia (14.4%, pâ¯=â¯0.044) and vomiting (60%, pâ¯<â¯0.001) than those receiving Act-d (5%, none, 2.5%, none, 10%, respectively). CONCLUSION: Carboplatin did not have a satisfactory clinical response rate, likely due to severe hematological toxicity, which postponed chemotherapy. Our results reinforce the preference for Act-d as a second-line agent in patients with low-risk GTN after MTX/FA resistance.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Substituição de Medicamentos , Doença Trofoblástica Gestacional/tratamento farmacológico , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/provisão & distribuição , Brasil , Carboplatina/farmacologia , Carboplatina/uso terapêutico , Dactinomicina/farmacologia , Dactinomicina/provisão & distribuição , Dactinomicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Etoposídeo/farmacologia , Etoposídeo/uso terapêutico , Feminino , Humanos , Metotrexato/farmacologia , Metotrexato/uso terapêutico , Gravidez , Estudos Retrospectivos , Resultado do Tratamento , Adulto JovemRESUMO
Increasing evidence indicates that the Golgi apparatus plays active roles in cancer, but a comprehensive understanding of its functions in the oncogenic transformation has not yet emerged. At the same time, the Golgi is becoming well recognized as a hub that integrates its functions of protein and lipid biosynthesis to signal transduction for cell proliferation and migration in cancer cells. Nevertheless, the active function of the Golgi apparatus in cancer cells has not been fully evaluated as a target for combined treatment. Here, we analyzed the effect of perturbing the Golgi apparatus on the sensitivity of the MDA-MB-231 breast cancer cell line to the drugs Actinomycin D and Vinblastine. We disrupted the function of ARF1, a protein necessary for the homeostasis of the Golgi apparatus. We found that the expression of the ARF1-Q71L mutant increased the sensitivity of MDA-MB-231 cells to both Actinomycin D and Vinblastine, resulting in decreased cell proliferation and cell migration, as well as in increased apoptosis. Likewise, the combined treatment of cells with Actinomycin D or Vinblastine and Brefeldin A or Golgicide A, two disrupting agents of the ARF1 function, resulted in similar effects on cell proliferation, cell migration and apoptosis. Interestingly, each combined treatment had distinct effects on ERK1/2 and AKT signaling, as indicated by the decreased levels of either phospho-ERK1/2 or phospho-AKT. Our results suggest that disruption of Golgi function could be used as a strategy for the sensitization of cancer cells to chemotherapy.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Dactinomicina/farmacologia , Vimblastina/farmacologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Wallerian degeneration is an active program tightly associated with axonal degeneration, required for axonal regeneration and functional recovery after nerve damage. Here we provide a functional molecular foundation for our undertstanding of the complex non-cell autonomous role of glial cells in the regulation of axonal degeneration. To shed light on the complexity of the molecular machinery governing axonal degeneration we employ a multi-model, unbiased, in vivo approach combining morphological assesment and quantitative proteomics with in silico-based higher order functional clustering to genetically uncouple the intrinsic and extrinsic processes governing Wallerian degeneration. Highlighting a pivotal role for glial cells in the early stages fragmenting the axon by a cytokinesis-like process and a cell autonomous stage of axonal disintegration associated to mitochondrial dysfunction.
Assuntos
Axônios/metabolismo , Neuroglia/metabolismo , Animais , Desdiferenciação Celular/efeitos dos fármacos , Células Cultivadas , Proteínas Contráteis/antagonistas & inibidores , Proteínas Contráteis/genética , Proteínas Contráteis/metabolismo , Peptidil-Prolil Isomerase F , Ciclofilinas/deficiência , Ciclofilinas/genética , Dactinomicina/farmacologia , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bainha de Mielina/fisiologia , Neuroglia/citologia , Proteômica , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/lesões , Degeneração Walleriana/metabolismo , Degeneração Walleriana/patologia , Quinases Associadas a rho/metabolismoRESUMO
Changes in RNA stability have an important impact in the gene expression regulation. Different methods based on the transcription blockage with RNA polymerase inhibitors or metabolic labeling of newly synthesized RNAs have been developed to evaluate RNA decay rates in cultured cell. Combined with techniques to measure transcript abundance genome-wide, these methods have been used to reveal novel features of the eukaryotic transcriptome. The stability of protein-coding mRNAs is in general closely associated to the physiological function of their encoded proteins, with short-lived mRNAs being significantly enriched among regulatory genes whereas genes associated with housekeeping functions are predominantly stable. Likewise, the stability of noncoding RNAs (ncRNAs) seems to reflect their functional role in the cell. Thus, investigating RNA stability can provide insights regarding the function of yet uncharacterized regulatory ncRNAs. In this chapter, we discuss the methodologies currently used to estimate RNA decay and outline an experimental protocol for genome-wide estimation of RNA stability of protein-coding and lncRNAs. This protocol details the transcriptional blockage of cultured cells with actinomycin D, followed by RNA isolation at different time points, the determination of transcript abundance by qPCR/DNA oligoarray hybridization, and the calculation of individual transcript half-lives.
Assuntos
RNA Mensageiro/química , RNA Mensageiro/isolamento & purificação , RNA não Traduzido/química , RNA não Traduzido/isolamento & purificação , Técnicas de Cultura de Células , Dactinomicina/farmacologia , Perfilação da Expressão Gênica , Genes Essenciais , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Estabilidade de RNA , Transcrição GênicaRESUMO
Tumor necrosis factor alpha (TNF-α) is a potent proinflammatory cytokine that plays a significant role in the pathogenesis of asthma by inducing hyperresponsiveness and airway remodeling. TNF-α diminishes the L-type voltage dependent Ca(2+) channel (L-VDCC) current in cardiac myocytes, an observation that seems paradoxical. In guinea pig sensitized tracheas KCl responses were lower than in control tissues. Serum from sensitized animals (Ser-S) induced the same phenomenon. In tracheal myocytes from nonsensitized (NS) and sensitized (S) guinea pigs, an L-VDCC current (ICa) was observed and diminished by Ser-S. The same decrease was detected in NS myocytes incubated with TNF-α, pointing out that this cytokine might be present in Ser-S. We observed that a small-molecule inhibitor of TNF-α (SMI-TNF) and a TNF-α receptor 1 (TNFR1) antagonist (WP9QY) reversed ICa decrease induced by Ser-S in NS myocytes, confirming the former hypothesis. U0126 (a blocker of ERK 1/2 kinase) also reverted the decrease in ICa. Neither cycloheximide (a protein synthesis inhibitor) nor actinomycin D (a transcription inhibitor) showed any effect on the TNF-α-induced ICa reduction. We found that CaV1.2 and CaV1.3 mRNA and proteins were expressed in tracheal myocytes and that sensitization did not modify them. In cardiac myocytes, ERK 1/2 phosphorylates two sites of the L-VDCC, augmenting or decreasing ICa; we postulate that, in guinea pig tracheal smooth muscle, TNF-α diminishes ICa probably by phosphorylating the L-VDCC site that reduces its activity through the ERK1/2 MAP kinase pathway.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Butadienos/farmacologia , Células Cultivadas , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Cobaias , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Nitrilas/farmacologia , Peptídeos Cíclicos/farmacologia , Traqueia/citologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Leishmania contains two phosphoglycerate kinase (PGK) genes, PGKB and PGKC, which code for the cytosolic and glycosomal isoforms of the enzyme, respectively. Although differences in PGKB and PGKC transcript and protein levels and isoform activities have been well documented, the mechanisms of control of both transcript and protein abundance have not been described to date. To better understand the regulation of Leishmania PGK expression, we investigated the stabilities of both PGK transcripts using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) in combination with transcription and trans-splicing inhibitors. Cells were treated with sinefungin and actinomycin D, and RNA decay kinetics were assessed. In addition, immunoblotting and protein synthesis inhibition by cycloheximide were employed to evaluate protein steady states and degradation. We observed increased stabilities of both PGKB mRNA and protein compared with the glycosomal isoform (PGKC). Our results indicate that both post-transcriptional and post-translational events contribute to the distinct expression levels of the PGKB and PGKC isoforms in Leishmania major.
Assuntos
Leishmania major/enzimologia , Fosfoglicerato Quinase/genética , Adenosina/análogos & derivados , Adenosina/farmacologia , Antiprotozoários/farmacologia , Cicloeximida/farmacologia , Citosol/enzimologia , Dactinomicina/farmacologia , Regulação da Expressão Gênica , Meia-Vida , Immunoblotting , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Leishmania major/efeitos dos fármacos , Leishmania major/genética , Microcorpos/enzimologia , Peso Molecular , Fosfoglicerato Quinase/química , Fosfoglicerato Quinase/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Long noncoding RNAs (lncRNAs) that map to intragenic regions of the human genome with the same (intronic lncRNAs) or opposite orientation (antisense lncRNAs) relative to protein-coding mRNAs have been largely dismissed from biochemical and functional characterization due to the belief that they are mRNA precursors, byproducts of RNA splicing or simply transcriptional noise. In this work, we used a custom microarray to investigate aspects of the biogenesis, processing, stability, evolutionary conservation, and cellular localization of â¼ 6,000 intronic lncRNAs and â¼ 10,000 antisense lncRNAs. Most intronic (2,903 of 3,427, 85%) and antisense lncRNAs (4,945 of 5,214, 95%) expressed in HeLa cells showed evidence of 5' cap modification, compatible with their transcription by RNAP II. Antisense lncRNAs (median t1/2 = 3.9 h) were significantly (p < 0.0001) more stable than mRNAs (median t1/2 = 3.2 h), whereas intronic lncRNAs (median t1/2 = 2.1 h) comprised a more heterogeneous class that included both stable (t1/2 > 3 h) and unstable (t1/2 < 1 h) transcripts. Intragenic lncRNAs display evidence of evolutionary conservation, have little/no coding potential and were ubiquitously detected in the cytoplasm. Notably, a fraction of the intronic and antisense lncRNAs (13 and 15%, respectively) were expressed from loci at which the corresponding host mRNA was not detected. The abundances of a subset of intronic/antisense lncRNAs were correlated (r ≥ |0.8|) with those of genes encoding proteins involved in cell division and DNA replication. Taken together, the findings of this study contribute novel biochemical and genomic information regarding intronic and antisense lncRNAs, supporting the notion that these classes include independently transcribed RNAs with potentials for exerting regulatory functions in the cell.
Assuntos
Perfilação da Expressão Gênica/métodos , Genoma Humano/genética , Íntrons/genética , RNA Antissenso/genética , RNA Longo não Codificante/genética , Alfa-Amanitina/farmacologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Dactinomicina/farmacologia , Decitabina , Células HeLa , Humanos , Células MCF-7 , Inibidores da Síntese de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , Estabilidade de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Thyroid hormone (TH) activates PI3K and Akt, leading to glucose uptake in rat skeletal muscle cells and proliferation of insulinoma cells, respectively. However, TH actions on pancreatic beta cells have been little explored, which lead us to evaluate the TH eff ects on proinsulin gene expression, and the involvement of PI3K/Akt/GSK-3ß signaling pathway, and a transcriptional factor for insulin (PDX-1). INS-1E cells were sorted into 3 groups: control and TH-depleted treated or not with T3 for 30 min. Cells were also previously treated with actinomycin D (ActD), cycloheximide (CHX), wortmannin or Akt inhibitor. Proinsulin mRNA expression was evaluated by real time PCR, and pGSK-3ß and PDX-1 protein content was analyzed by Western blotting. TH depletion decreased proinsulin mRNA content, which was restored after acute T3 treatment. ActD, CHX and wortmannin, but not Akt inhibitor, prevented the rapid stimulatory eff ect of T3 on proinsulin mRNA expression. TH depletion did not affect the phosphorylated GSK-3ß and PDX-1 protein content; but T3 treatment led to an increase in the content of these proteins. These data indicate that T3 acutely increases proinsulin mRNA expression, by mechanisms which depends on the activation of PI3K, but not of Akt, and may involve the inactivation of GSK-3ß by phosphorylation. Since GSK-3ß enhances PDX-1 degradation rate, the GSK-3ß inactivation could explain the increase of PDX-1 content in T3-treated cells. Considering that PDX-1 is one of the most important transcriptional factors for proinsulin gene expression, its enhancement may underlie the increased proinsulin mRNA content acutely induced by T3.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas de Homeodomínio/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proinsulina/biossíntese , Transativadores/metabolismo , Tri-Iodotironina/farmacologia , Animais , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Regulação da Expressão Gênica/genética , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Proteínas de Homeodomínio/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proinsulina/genética , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Transativadores/genéticaRESUMO
Multidrug resistance-associated protein 3 (Mrp3; Abcc3) expression and activity are up-regulated in rat liver after in vivo repeated administration of ethynylestradiol (EE), a cholestatic synthetic estrogen, whereas multidrug resistance-associated protein 2 (Mrp2) is down-regulated. This study was undertaken to determine whether Mrp3 induction results from a direct effect of EE, independent of accumulation of any endogenous common Mrp2/Mrp3 substrates resulting from cholestasis and the potential mediation of estrogen receptor (ER). In in vivo studies, male rats were given a single, noncholestatic dose of EE (5 mg/kg s.c.), and basal bile flow and the biliary excretion rate of bile salts and glutathione were measured 5 hours later. This treatment increased Mrp3 mRNA by 4-fold, detected by real-time polymerase chain reaction, despite the absence of cholestasis. Primary culture of rat hepatocytes incubated with EE (1-10 µM) for 5 hours exhibited a 3-fold increase in Mrp3 mRNA (10 µM), consistent with in vivo findings. The increase in Mrp3 mRNA by EE was prevented by actinomycin D, indicating transcriptional regulation. When hepatocytes were incubated with an ER antagonist [7α,17ß-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI182/780), 1 µM], in addition to EE, induction of Mrp3 mRNA was abolished, implicating ER as a key mediator. EE induced an increase in ER-α phosphorylation at 30 minutes and expression of c-Jun, a well-known ER target gene, at 60 minutes, as detected by Western blotting of nuclear extracts. These increases were prevented by ICI182/780. In summary, EE increased the expression of hepatic Mrp3 transcriptionally and independently of any cholestatic manifestation and required participation of an ER, most likely ER-α, through its phosphorylation.