RESUMEN
Bio-guided fractionation performed on the leaves-derived ethanol extract of Esenbeckia alata (Rutaceae), a plant used in traditional medicine, led to the isolation of two alkaloids, kokusaginine 1 and flindersiamine 2, as main cytotoxic agents. Primary ethanolic extract and raw fractions exhibited cell inhibition against five cancer cell lines at different levels (25-97% inhibition at 50 µg/mL) as well as isolated alkaloids 1-2 (30-90% inhibition at 20 µM). Although alkaloid 2 generally was the most active compound, both alkaloids showed a selective effect on K562, a human chronic myelogenous leukemia cell line. The E1-like ubiquitin-activating enzymes (e.g., UBA5) have been recently described as important targets for future treatment of cancer progression, such as leukemia, among others. Therefore, as a rationale to the observed cytotoxic selectivity, an in-silico evaluation by molecular docking and molecular dynamics was also explored. Compounds 1-2 exhibited good performance on the interaction within the active site of UBA5.
Asunto(s)
Alcaloides/química , Antineoplásicos Fitogénicos/química , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Quinolinas/química , Rutaceae/química , Enzimas Activadoras de Ubiquitina/metabolismo , Alcaloides/farmacología , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fraccionamiento Químico , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Extractos Vegetales/química , Hojas de la Planta/química , Quinolinas/farmacología , Enzimas Activadoras de Ubiquitina/químicaRESUMEN
The use of carboplatin in cancer chemotherapy is limited by the emergence of drug resistance. To understand the molecular basis for this resistance, a chemogenomic screen was performed in 53 yeast mutants that had previously presented strong sensitivity to this widely used anticancer agent. Thirty-four mutants were responsive to carboplatin, and from these, 21 genes were selected for further studies because they have human homologues. Sixty percent of these yeast genes possessed human homologues which encoded proteins that interact with cullin scaffolds of ubiquitin ligases, or whose mRNA are under the regulation of Human antigen R (HuR) protein. Both HuR and cullin proteins are regulated through NEDDylation post-translational modification, and so our results indicate that inhibition of this process should sensitise resistant tumour cells to carboplatin. We showed that treatment of a tumour cell line with MLN4924, a NEDDylation inhibitor, overcame the resistance to carboplatin. Our data suggest that inhibition of NEDDylation may be a useful strategy to resensitise tumour cells in patients that have acquired carboplatin resistance.
Asunto(s)
Carboplatino/farmacología , Proteínas Cullin/genética , Resistencia a Antineoplásicos/genética , Proteína 1 Similar a ELAV/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Línea Celular Tumoral/efectos de los fármacos , Cromosomas Humanos Par 1 , Proteínas Cullin/metabolismo , Ciclopentanos/farmacología , Farmacorresistencia Fúngica/efectos de los fármacos , Farmacorresistencia Fúngica/fisiología , Resistencia a Antineoplásicos/efectos de los fármacos , Proteína 1 Similar a ELAV/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Mutación , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Pirimidinas/farmacología , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Enzimas Activadoras de Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/metabolismoRESUMEN
Giardia intestinalis is considered an early-branching eukaryote and is therefore a valuable model for studying primordial cellular processes. This work reports the characterization of the ubiquitin-activating enzyme (E1) during growth and different stages of trophozoite differentiation into cysts. We found that in Giardia E1 expression (both at mRNA and protein levels) is regulated during encystation. The enzyme is proteolytically processed mainly into two fragments of 68kDa (N-terminal) and 47kDa (C-terminal). This phenomenon has not been described for any other E1. In trophozoites, this enzyme localized at spots within the cytoplasm as detected by using polyclonal antibodies against either E1 N- or C-terminal fragments. This pattern changed during encystation into a diffuse localization throughout the cytoplasm of encysting cells. E1 localizes in mature cysts at cytoplasmic spots and in the cyst wall. Our antisense silencing experiments suggested that E1 is an essential gene for parasite viability. On the other hand, E1 over-expression greatly increased the encystation rate, indicating a relationship between E1 and Giardia differentiation.
Asunto(s)
Giardia lamblia/enzimología , Giardia lamblia/crecimiento & desarrollo , Enzimas Activadoras de Ubiquitina/metabolismo , Animales , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos BALB C , Viabilidad Microbiana , Proteolisis , Esporas Protozoarias/enzimología , Esporas Protozoarias/crecimiento & desarrollo , Trofozoítos/enzimología , Trofozoítos/crecimiento & desarrolloRESUMEN
Giardia intestinalis is a single-cell eukaryotic microorganism, regarded as one of the earliest divergent eukaryotes and thus an attractive model to study the evolution of regulatory systems. Giardia has two different forms throughout its life cycle, cyst and trophozoite, and changes from one to the other in response to environmental signals. The two differentiation processes involve a differential gene expression as well as a quick and specific protein turnover that may be mediated by the ubiquitin/proteasome system. The aim of this work was to search for unreported components of the ubiquitination system and to experimentally demonstrate their expression in the parasite and during the two differentiation processes. We found activity of protein ubiquitination in G. intestinalis trophozoites and analyzed the transcription of the ubiquitin gene, as well as that of the activating (E1), conjugating (E2), and ligase (E3) ubiquitin enzymes during encystation and excystation. A constant ubiquitin expression persisted during the parasite's differentiation processes, whereas variation in transcription was observed in the other genes under study.