RESUMO
The aim of this study was to evaluate the cytotoxic potential of Aristolochia foetida Kunth. Stems and leaves of A. foetida Kunth (Aristolochiaceae) have never been investigated pharmacologically. Recent studies of species of the Aristolochiaceae family found significant cytotoxic activities. Hexane, dichloromethane, ethyl acetate and methanol extracts were analyzed by 1H NMR and GC-MS to know the metabolites in each extract. In GC-MS analysis, the main compounds were methyl hexadecanoate (3); hexadecanoic acid (4); 2-butoxyethyl dodecanoate (9); ethyl hexadecanoate (20); methyl octadeca-9,12,15-trienoate (28) and (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid (40). The results showed a significant reduction in cell viability of the MCF-7 (breast cancer) cell line caused by organic extracts in a dose-dependent manner. The cytotoxicity activity of the dichloromethane extract from the stems (DSE) showed IC50 values of 45.9 µg/mL and the dichloromethane extract of the leaves (DLE) showed IC50 values of 47.3 µg/mL. DSE and DLE had the highest cytotoxic potential in an in vitro study against the MCF-7 cell line and non-tumor cells obtained from the bovine mammary epithelial (bMECs). DSE and DLE induced a loss in mitochondrial membrane potential (ΔΨm) and can cause cell death by apoptosis through the intrinsic pathway in the MCF-7 cell line. DSE and DLE are cytotoxic in cancer cells and cause late apoptosis. Higher concentrations of DSE and DLE are required to induce a cytotoxic effect in healthy mammary epithelial cells. This is the first report of the dichloromethane extract of A. foetida Kunth that induces late apoptosis in MCF-7 cancer cells and may be a candidate for pharmacological study against breast cancer.
RESUMO
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
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
High amounts of albumin in urine cause tubulointerstitial damage that leads to a rapid deterioration of the renal function. Albumin exerts its injurious effects on renal cells through a process named endoplasmic reticulum (ER) stress due to the accumulation of unfolded proteins in the ER lumen. In addition, albumin promotes phosphorylation and consequent activation of MAPKs such as ERK1/2. Since ERK1/2 activation promoted by albumin is a transient event, the aims of the present work were to identify the phosphatase involved in their dephosphorylation in albumin-exposed cells and to analyze the putative regulation of this phosphatase by albumin. We also sought to determine the role played by the phospho/dephosphorylation of ERK1/2 in the cellular response to albumin-induced ER stress. MAP kinase phosphatase-1, MKP-1, is a nuclear enzyme involved in rapid MAPK dephosphorylation. Here we present evidence supporting the notion that this phosphatase is responsible for ERK1/2 dephosphorylation after albumin exposure in OK cells. Moreover, we demonstrate that exposure of OK cells to albumin transiently increases MKP-1 protein levels. The increase was evident after 15 min of exposure, peaked at 1 h (6-fold) and declined thereafter. In cells overexpressing flag-MKP-1, albumin caused the accumulation of this chimera, promoting MKP-1 stabilization by a posttranslational mechanism. Albumin also promoted a transient increase in MKP-1 mRNA levels (3-fold at 1 h) through the activation of gene transcription. In addition, we also show that albumin increased mRNA levels of GRP78, a key marker of ER stress, through an ERK-dependent pathway. In line with this finding, our studies demonstrate that flag-MKP-1 overexpression blunted albumin-induced GRP78 upregulation. Thus, our work demonstrates that albumin overload not only triggers MAPK activation but also tightly upregulates MKP-1 expression, which might modulate ER stress response to albumin overload.
Assuntos
Didelphis/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , Retículo Endoplasmático/metabolismo , Túbulos Renais Proximais/metabolismo , Estresse Oxidativo , Soroalbumina Bovina/metabolismo , Animais , Bovinos , Células Cultivadas , Fosfatase 1 de Especificidade Dupla/genética , Túbulos Renais Proximais/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
An actinomycin-D producing strain was isolated from soil and characterized as Streptomyces sindenensis. The culture was subjected to UV irradiation and a mutant with 400 percent higher actinomycin-D production was isolated (400 mg/l-1 as compared to 80 mg/l-1 produced by the parent). Production medium was optimized and antibiotic yield with the mutant was enhanced to 850 mg/l-1 which is 963 percent higher as compared with the parent.
Uma cepa produtora de actinomicina-D foi isolada de solo e caracterizada como Streptomyces sindenensis. A cultura foi submetida à radiação UV, e um mutante capaz de produzir 400 por cento mais actinomicina-D foi isolado (400mg/L comparado a 80mg/L produzido pela cepa parental). O meio de produção do antibiótico foi otimizado e o rendimento aumentou para 850 mg/L, ou seja, 963 por cento mais alto que a cepa parental.
Assuntos
Antibacterianos/isolamento & purificação , Dactinomicina/isolamento & purificação , Mutagênicos , Radiação , Streptomyces antibioticus/isolamento & purificação , Métodos , Solo , MétodosRESUMO
An actinomycin-D producing strain was isolated from soil and characterized as Streptomyces sindenensis. The culture was subjected to UV irradiation and a mutant with 400% higher actinomycin-D production was isolated (400 mg/l(-1) as compared to 80 mg/l(-1) produced by the parent). Production medium was optimized and antibiotic yield with the mutant was enhanced to 850 mg/l(-1) which is 963% higher as compared with the parent.
RESUMO
An actinomycin-D producing strain was isolated from soil and characterized as Streptomyces sindenensis. The culture was subjected to UV irradiation and a mutant with 400% higher actinomycin-D production was isolated (400 mg/l-1 as compared to 80 mg/l-1 produced by the parent). Production medium was optimized and antibiotic yield with the mutant was enhanced to 850 mg/l-1 which is 963% higher as compared with the parent.
Uma cepa produtora de actinomicina-D foi isolada de solo e caracterizada como Streptomyces sindenensis. A cultura foi submetida à radiação UV, e um mutante capaz de produzir 400% mais actinomicina-D foi isolado (400mg/L comparado a 80mg/L produzido pela cepa parental). O meio de produção do antibiótico foi otimizado e o rendimento aumentou para 850 mg/L, ou seja, 963% mais alto que a cepa parental.