RESUMO
The antiproliferative activity of [Mn(CO)3(N^N)Br] (N^N = phendione 1, bipy 3) and of the two newly synthesized Mn complexes [Mn(CO)3(acridine)(phendione)]OTf (2) and [Mn(CO)3(di-triazole)Br] (4) has been evaluated by MTS against three tumor cell lines A2780 (ovarian carcinoma), HCT116 (colorectal carcinoma), HCT116doxR (colorectal carcinoma resistant to doxorubicin), and in human dermal fibroblasts. The antiproliferative assay showed a dose-dependent effect higher in complex 1 and 2 with a selectivity toward ovarian carcinoma cell line 21 times higher than in human fibroblasts. Exposure of A2780 cells to IC50 concentrations of complex 1 and 2 led to an increase of reactive oxygen species that led to the activation of cell death mechanisms, namely via intrinsic apoptosis for 2 and autophagy and extrinsic apoptosis for 1. Both complexes do not target DNA or interfere with cell cycle progression but are able to potentiate cell migration and neovascularization (for 2) an indicative that their application might be directed for initial tumor stages to avoid tumor invasion and metastization and opening a new avenue for complex 2 application in regenerative medicine. Interestingly, both complexes do not show toxicity in both in vivo models (CAM and zebrafish).
Assuntos
Antineoplásicos , Complexos de Coordenação , Neoplasias Ovarianas , Animais , Antineoplásicos/química , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Complexos de Coordenação/química , Feminino , Humanos , Manganês , Neoplasias Ovarianas/patologia , Peixe-ZebraRESUMO
The human cancer cell xenograft in zebrafish embryos has become a very useful preclinical tool in oncology research. While many anticancer drugs have been assayed with this model, few studies regarding the toxicity limits of these drugs for the host have been addressed. Here, we evaluated the acute toxicity of five approved and routinely used human anticancer drugs embracing different mechanism action types: Carboplatin (CarboPt), Irinotecan (IT), Doxorubicin (DOX), Paclitaxel (PT) and Chloroquine (CQ). They were tested in zebrafish embryos using the Fish Embryo Acute Toxicity (FET) test at 0 and 72 hours per fertilization (hpf). Additionally, we compared those results with in vitro toxicity assays and could find notable differences between both models. Our results indicate that the toxicity data of a compound evaluated in vitro and in a FET test at 0 hpf do not guarantee a reliable toxicity determination for performing xenografts in zebrafish, being necessary additional toxicity studies using 72 hpf embryos, the starting point of drug treatment in this kind of preclinical assays.
Assuntos
Antineoplásicos/toxicidade , Avaliação Pré-Clínica de Medicamentos/métodos , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra , Animais , Fatores de Tempo , Testes de Toxicidade AgudaRESUMO
BACKGROUND: Zebrafish (Danio rerio) is a model organism that has emerged as a tool for cancer research, cancer being the second most common cause of death after cardiovascular disease for humans in the developed world. Zebrafish is a useful model for xenotransplantation of human cancer cells and toxicity studies of different chemotherapeutic compounds in vivo. Compared to the murine model, the zebrafish model is faster, can be screened using high-throughput methods and has a lower maintenance cost, making it possible and affordable to create personalized therapies. While several methods for cell proliferation determination based on image acquisition and quantification have been developed, some drawbacks still remain. In the xenotransplantation technique, quantification of cellular proliferation in vivo is critical to standardize the process for future preclinical applications of the model. METHODS: This study improved the conditions of the xenotransplantation technique - quantification of cellular proliferation in vivo was performed through image processing with our ZFtool software and optimization of temperature in order to standardize the process for a future preclinical applications. ZFtool was developed to establish a base threshold that eliminates embryo auto-fluorescence and measures the area of marked cells (GFP) and the intensity of those cells to define a 'proliferation index'. RESULTS: The analysis of tumor cell proliferation at different temperatures (34 °C and 36 °C) in comparison to in vitro cell proliferation provides of a better proliferation rate, achieved as expected at 36°, a maintenance temperature not demonstrated up to now. The mortality of the embryos remained between 5% and 15%. 5- Fluorouracil was tested for 2 days, dissolved in the incubation medium, in order to quantify the reduction of the tumor mass injected. In almost all of the embryos incubated at 36 °C and incubated with 5-Fluorouracil, there was a significant tumor cell reduction compared with the control group. This was not the case at 34 °C. CONCLUSIONS: Our results demonstrate that the proliferation of the injected cells is better at 36 °C and that this temperature is the most suitable for testing chemotherapeutic drugs like the 5-Fluorouracil.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Embrião não Mamífero/citologia , Proteínas de Fluorescência Verde/metabolismo , Neoplasias/diagnóstico , Software , Animais , Modelos Animais de Doenças , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Temperatura , Transplante Heterólogo , Células Tumorais Cultivadas , Peixe-ZebraRESUMO
Ciguatoxins are sodium channels activators that cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents with long-term neurological alterations. In central neurons, chronic perturbations in activity induce homeostatic synaptic mechanisms that adjust the strength of excitatory synapses and modulate glutamate receptor expression in order to stabilize the overall activity. Immediate early genes, such as Arc and Egr1, are induced in response to activity changes and underlie the trafficking of glutamate receptors during neuronal homeostasis. To better understand the long lasting neurological consequences of ciguatera, it is important to establish the role that chronic changes in activity produced by ciguatoxins represent to central neurons. Here, the effect of a 30 min exposure of 10-13 days in vitro (DIV) cortical neurons to the synthetic ciguatoxin CTX 3C on Arc and Egr1 expression was evaluated using real-time polymerase chain reaction approaches. Since the toxin increased the mRNA levels of both Arc and Egr1, the effect of CTX 3C in NaV channels, membrane potential, firing activity, miniature excitatory postsynaptic currents (mEPSCs), and glutamate receptors expression in cortical neurons after a 24 h exposure was evaluated using electrophysiological and western blot approaches. The data presented here show that CTX 3C induced an upregulation of Arc and Egr1 that was prevented by previous coincubation of the neurons with the NaV channel blocker tetrodotoxin. In addition, chronic CTX 3C caused a concentration-dependent shift in the activation voltage of NaV channels to more negative potentials and produced membrane potential depolarization. Moreover, 24 h treatment of cortical neurons with 5 nM CTX 3C decreased neuronal firing and induced synaptic scaling mechanisms, as evidenced by a decrease in the amplitude of mEPSCs and downregulation in the protein level of glutamate receptors that was also prevented by tetrodotoxin. These findings identify an unanticipated role for ciguatoxin in the regulation of homeostatic plasticity in central neurons involving NaV channels and raise the possibility that some of the neurological symptoms of ciguatera might be explained by these compensatory mechanisms.
Assuntos
Córtex Cerebral/citologia , Ciguatoxinas/toxicidade , Neurônios/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Ciguatoxinas/administração & dosagem , Relação Dose-Resposta a Droga , Camundongos , Neurônios/metabolismo , Relação Estrutura-Atividade , Sinapses/metabolismoRESUMO
The Mediterranean marine sponge Crambe crambe is the source of two families of guanidine alkaloids known as crambescins and crambescidins. Some of the biological effects of crambescidins have been previously reported while crambescins have undergone little study. Taking this into account, we performed comparative transcriptome analysis to examine the effect of crambescin-C1 (CC1) on human tumor hepatocarcinoma cells HepG2 followed by validation experiments to confirm its predicted biological activities. We report herein that, while crambescin-A1 has a minor effect on these cells, CC1 protects them against oxidative injury by means of metallothionein induction even at low concentrations. Additionally, at high doses, CC1 arrests the HepG2 cell cycle in G0/G1 and thus inhibits tumor cell proliferation. The findings presented here provide the first detailed approach regarding the different effects of crambescins on tumor cells and provide a basis for future studies on other possible cellular mechanisms related to these bioactivities.
Assuntos
Fatores Biológicos/farmacologia , Citoproteção/efeitos dos fármacos , Metalotioneína/metabolismo , Pirimidinas/farmacologia , Alcaloides/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Crambe (Esponja)/metabolismo , Fase G1/efeitos dos fármacos , Células Hep G2 , Humanos , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Transcriptoma/efeitos dos fármacosRESUMO
BACKGROUND: Previous studies by our group have shown that oxidative phosphorylation (OXPHOS) is the main pathway by which pancreatic cancer stem cells (CSCs) meet their energetic requirements; therefore, OXPHOS represents an Achille's heel of these highly tumorigenic cells. Unfortunately, therapies that target OXPHOS in CSCs are lacking. METHODS: The safety and anti-CSC activity of a ruthenium complex featuring bipyridine and terpyridine ligands and one coordination labile position (Ru1) were evaluated across primary pancreatic cancer cultures and in vivo, using 8 patient-derived xenografts (PDXs). RNAseq analysis followed by mitochondria-specific molecular assays were used to determine the mechanism of action. RESULTS: We show that Ru1 is capable of inhibiting CSC OXPHOS function in vitro, and more importantly, it presents excellent anti-cancer activity, with low toxicity, across a large panel of human pancreatic PDXs, as well as in colorectal cancer and osteosarcoma PDXs. Mechanistic studies suggest that this activity stems from Ru1 binding to the D-loop region of the mitochondrial DNA of CSCs, inhibiting OXPHOS complex-associated transcription, leading to reduced mitochondrial oxygen consumption, membrane potential, and ATP production, all of which are necessary for CSCs, which heavily depend on mitochondrial respiration. CONCLUSIONS: Overall, the coordination complex Ru1 represents not only an exciting new anti-cancer agent, but also a molecular tool to dissect the role of OXPHOS in CSCs. Results indicating that the compound is safe, non-toxic and highly effective in vivo are extremely exciting, and have allowed us to uncover unprecedented mechanistic possibilities to fight different cancer types based on targeting CSC OXPHOS.
Assuntos
Neoplasias Pancreáticas , Rutênio , Humanos , Fosforilação Oxidativa , Rutênio/farmacologia , Mitocôndrias/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Células-Tronco Neoplásicas/metabolismoRESUMO
In this paper, we show the effect of crambescidin-816, -800, and -830 on Saccharomyces cerevisiae viability. We determined that, of the three molecules tested, crambescidin-816 was the most potent. Based on this result, we continued by determining the effect of crambescidin-816 on the cell cycle of this yeast. The compound induced cell cycle arrest in G2/M followed by an increase in cell DNA content and size. When the type of cell death was analyzed, we observed that crambescidin-816 induced apoptosis. The antifungal effect indicates that crambescidins, and mostly crambescidin-816, could serve as a lead compound to fight fungal infections.
Assuntos
Alcaloides/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Fungicidas Industriais/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Compostos de Espiro/farmacologia , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Guanidina/análogos & derivados , Guanidina/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacosRESUMO
Metamorphosis is a captivating process of change during which the morphology of the larva is completely reshaped to face the new challenges of adult life. In the case of fish, this process initiated in the brain has traditionally been considered to be a critical rearing point and despite the pioneering molecular work carried out in other flatfishes, the underlying molecular basis is still relatively poorly characterized. Turbot brain transcriptome of three developmental stages (pre-metamorphic, climax of metamorphosis and post-metamorphic) were analyzed to study the gene expression dynamics throughout the metamorphic process. A total of 1570 genes were differentially expressed in the three developmental stages and we found a specific pattern of gene expression at each stage. Unexpectedly, at the climax stage of metamorphosis, we found highly expressed genes related to the immune response, while the biological pathway enrichment analysis in pre-metamorphic and post-metamorphic were related to cell differentiation and oxygen carrier activity, respectively. In addition, our results confirm the importance of thyroid stimulating hormone, increasing its expression during metamorphosis. Based on our findings, we assume that immune system activation during the climax of metamorphosis stage could be related to processes of larval tissue inflammation, resorption and replacement, as occurs in other vertebrates.
RESUMO
The neural crest is a multipotent cell population that develops from the dorsal neural fold of vertebrate embryos in order to migrate extensively and differentiate into a variety of tissues. A number of gene regulatory networks coordinating neural crest cell specification and differentiation have been extensively studied to date. Although several publications suggest a common role for microRNA-145 (miR-145) in molecular reprogramming for cell cycle regulation and/or cellular differentiation, little is known about its role during in vivo cranial neural crest development. By modifying miR-145 levels in zebrafish embryos, abnormal craniofacial development and aberrant pigmentation phenotypes were detected. By whole-mount in situ hybridization, changes in expression patterns of col2a1a and Sry-related HMG box (Sox) transcription factors sox9a and sox9b were observed in overexpressed miR-145 embryos. In agreement, zebrafish sox9b expression was downregulated by miR-145 overexpression. In silico and in vivo analysis of the sox9b 3'UTR revealed a conserved potential miR-145 binding site likely involved in its post-transcriptional regulation. Based on these findings, we speculate that miR-145 participates in the gene regulatory network governing zebrafish chondrocyte differentiation by controlling sox9b expression.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , MicroRNAs/genética , Crista Neural/citologia , Organogênese , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Animais , Diferenciação Celular , Anormalidades Craniofaciais/etiologia , Anormalidades Craniofaciais/metabolismo , Anormalidades Craniofaciais/patologia , Crista Neural/metabolismo , Transtornos da Pigmentação/etiologia , Transtornos da Pigmentação/metabolismo , Transtornos da Pigmentação/patologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Published studies show that most of the human cancer xenograft studies in zebrafish embryos have used incubation temperatures in the range of 32-34 °C for 3-6 days post-injection, trying to find a compromise temperature between the zebrafish embryos (28 °C) and the human injected cells (37 °C). While this experimental setup is widely used, a question remains: is possible to overcome the drawbacks caused by a suboptimal temperature for the injected cells? To clarify the effect of temperature and injected cells on the host, in this study, we analyzed the development and health of the last in response to different temperatures in the presence or absence of injected human cancer cells. Comparing different incubation temperatures (28, 34 and 36 °C), we determined morphological abnormalities and developmental effects in injected and non-injected embryos at different time points. Besides this, the expression of selected genes was determined by qPCR to determine temperature affected metabolic processes in the embryos. The results indicate that an incubation temperature of 36 °C during a period of 48 h is suitable for xenotransplantation without morphological or metabolic changes that could be affecting the host or the injected cells, allowing them to proliferate near their optimal temperature.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/imunologia , Temperatura Alta/efeitos adversos , Neoplasias/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Peixe-Zebra/fisiologia , Animais , Linhagem Celular Tumoral/fisiologia , Linhagem Celular Tumoral/transplante , Proliferação de Células/genética , Embrião não Mamífero/fisiologia , Humanos , Imunidade Inata/genética , Neoplasias/patologia , Especificidade da EspécieRESUMO
Crambescins are guanidine alkaloids from the sponge Crambe crambe. Crambescin C1 (CC) induces metallothionein genes and nitric oxide (NO) is one of the triggers. We studied and compared the in vitro, in vivo, and in silico effects of some crambescine A and C analogs. HepG2 gene expression was analyzed using microarrays. Vasodilation was studied in rat aortic rings. In vivo hypotensive effect was directly measured in anesthetized rats. The targets of crambescines were studied in silico. CC and homo-crambescine C1 (HCC), but not crambescine A1 (CA), induced metallothioneins transcripts. CC increased NO production in HepG2 cells. In isolated rat aortic rings, CC and HCC induced an endothelium-dependent relaxation related to eNOS activation and an endothelium-independent relaxation related to iNOS activation, hence both compounds increase NO and reduce vascular tone. In silico analysis also points to eNOS and iNOS as targets of Crambescin C1 and source of NO increment. CC effect is mediated through crambescin binding to the active site of eNOS and iNOS. CC docking studies in iNOS and eNOS active site revealed hydrogen bonding of the hydroxylated chain with residues Glu377 and Glu361, involved in the substrate recognition, and explains its higher binding affinity than CA. The later interaction and the extra polar contacts with its pyrimidine moiety, absent in the endogenous substrate, explain its role as exogenous substrate of NOSs and NO production. Our results suggest that CC serve as a basis to develop new useful drugs when bioavailability of NO is perturbed.
RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors, partly due to its intrinsic aggressiveness, metastatic potential, and chemoresistance of the contained cancer stem cells (CSCs). Pancreatic CSCs strongly rely on mitochondrial metabolism to maintain their stemness, therefore representing a putative target for their elimination. Since mitochondrial homeostasis depends on the tightly controlled balance between fusion and fission processes, namely mitochondrial dynamics, we aim to study this mechanism in the context of stemness. In human PDAC tissues, the mitochondrial fission gene DNM1L (DRP1) was overexpressed and positively correlated with the stemness signature. Moreover, we observe that primary human CSCs display smaller mitochondria and a higher DRP1/MFN2 expression ratio, indicating the activation of the mitochondrial fission. Interestingly, treatment with the DRP1 inhibitor mDivi-1 induced dose-dependent apoptosis, especially in CD133+ CSCs, due to the accumulation of dysfunctional mitochondria and the subsequent energy crisis in this subpopulation. Mechanistically, mDivi-1 inhibited stemness-related features, such as self-renewal, tumorigenicity, and invasiveness and chemosensitized the cells to the cytotoxic effects of Gemcitabine. In summary, mitochondrial fission is an essential process for pancreatic CSCs and represents an attractive target for designing novel multimodal treatments that will more efficiently eliminate cells with high tumorigenic potential.
RESUMO
Advances in transgenic technology as well as in the genetics of Alzheimer disease (AD) have allowed the establishment of animal models that reproduce amyloid-beta plaques and neurofibrillary tangles, the main pathological hallmarks of AD. Among these models, 3xTg-AD mice harboring PS1 (M146V), APP (Swe) and tau (P301L) human transgenes provided the model that most closely mimics human AD features. Although cortical cultures from 3xTg-AD mice have been shown to present disturbances in intracellular [Ca(2+)] homeostasis, the development of AD pathology in vitro has not been previously evaluated. In the current work, we determined the temporal profile for amyloid precursor protein, amyloid-beta and tau expression in primary cortical cultures from 3xTg-AD mice. Immunocytochemistry and Western blot analysis showed an increased expression of these proteins as well as several phosphorylated tau isoforms with time in culture. Alterations in calcium homeostasis and cholinergic and glutamatergic responses were also observed early in vitro. Thus, 3x-TgAD cortical neurons in vitro provide an exceptional tool to investigate pharmacological approaches as well as the cellular basis for AD and related diseases.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Córtex Cerebral/citologia , Neurônios/metabolismo , Proteínas tau/metabolismo , Acetilcolina/metabolismo , Doença de Alzheimer/patologia , Animais , Cálcio/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Ácido Glutâmico/metabolismo , Homeostase , Humanos , Camundongos , Camundongos Transgênicos , Neurônios/citologia , FosforilaçãoRESUMO
Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer death, has a 5-year survival rate of approximately 7-9%. The ineffectiveness of anti-PDAC therapies is believed to be due to the existence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are functionally plastic, and have exclusive tumorigenic, chemoresistant and metastatic capacities. Herein, we describe a 2D in vitro system for long-term enrichment of pancreatic CSCs that is amenable to biological and CSC-specific studies. By changing the carbon source from glucose to galactose in vitro, we force PDAC cells to utilize OXPHOS, resulting in enrichment of CSCs defined by increased CSC biomarker and pluripotency gene expression, greater tumorigenic potential, induced but reversible quiescence, increased OXPHOS activity, enhanced invasiveness, and upregulated immune evasion properties. This CSC enrichment method can facilitate the discovery of new CSC-specific hallmarks for future development into targets for PDAC-based therapies.
Assuntos
Carcinoma Ductal Pancreático/imunologia , Evasão da Resposta Imune , Células-Tronco Neoplásicas/imunologia , Neoplasias Pancreáticas/imunologia , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Feminino , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Células-Tronco Neoplásicas/metabolismo , Fosforilação Oxidativa , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologiaRESUMO
Growth is among the most important traits for animal breeding. Understanding the mechanisms underlying growth differences between individuals can contribute to improving growth rates through more efficient breeding schemes. Here, we report a transcriptomic study in muscle and brain of fast- and slow-growing turbot (Scophthalmus maximus), a relevant flatfish in European and Asian aquaculture. Gene expression and allelic association between the two groups were explored. Up-regulation of the anaerobic glycolytic pathway in the muscle of fast-growing fish was observed, indicating a higher metabolic rate of white muscle. Brain expression differences were smaller and not associated with major growth-related genes, but with regulation of feeding-related sensory pathways. Further, SNP variants showing frequency differences between fast- and slow-growing fish pointed to genomic regions likely involved in growth regulation, and three of them were individually validated through SNP typing. Although different mechanisms appear to explain growth differences among families, general mechanisms seem also to be involved, and thus, results provide a set of useful candidate genes and markers to be evaluated for more efficient growth breeding programs and to perform comparative genomic studies of growth in fish and vertebrates.
Assuntos
Encéfalo/metabolismo , Linguados/genética , Perfilação da Expressão Gênica , Desenvolvimento Muscular/genética , Polimorfismo de Nucleotídeo Único , Animais , Aquicultura/métodos , Encéfalo/crescimento & desenvolvimento , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Linguados/crescimento & desenvolvimento , Linguados/metabolismo , Frequência do Gene , Genômica/métodos , Glicólise/genética , FenótipoRESUMO
Flatfish have a high market acceptance thus representing a profitable aquaculture production. The main farmed species is the turbot (Scophthalmus maximus) followed by Japanese flounder (Paralichthys olivaceous) and tongue sole (Cynoglossus semilaevis), but other species like Atlantic halibut (Hippoglossus hippoglossus), Senegalese sole (Solea senegalensis) and common sole (Solea solea) also register an important production and are very promising for farming. Important genomic resources are available for most of these species including whole genome sequencing projects, genetic maps and transcriptomes. In this work, we integrate all available genomic information of these species within a common framework, taking as reference the whole assembled genomes of turbot and tongue sole (>210× coverage). New insights related to the genetic basis of productive traits and new data useful to understand the evolutionary origin and diversification of this group were obtained. Despite a general 1:1 chromosome syntenic relationship between species, the comparison of turbot and tongue sole genomes showed huge intrachromosomic reorganizations. The integration of available mapping information supported specific chromosome fusions along flatfish evolution and facilitated the comparison between species of previously reported genetic associations for productive traits. When comparing transcriptomic resources of the six species, a common set of ~2500 othologues and ~150 common miRNAs were identified, and specific sets of putative missing genes were detected in flatfish transcriptomes, likely reflecting their evolutionary diversification.
Assuntos
Aquicultura , Evolução Molecular , Linguados/genética , Genoma/genética , MicroRNAs/genética , Locos de Características Quantitativas , Transcriptoma , Animais , Biologia Computacional , Perfilação da Expressão Gênica , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Anotação de Sequência Molecular , FilogeniaRESUMO
In 2012, Tetrodotoxin (TTX) was identified in mussels and linked to the presence of Prorocentrum minimum (P. minimum) in Greece. The connexion between TTX and P. minimum was further studied in this paper. First, the presence of TTX-producer bacteria, Vibrio and Pseudomonas spp, was confirmed in Greek mussels. In addition these samples showed high activity as inhibitors of sodium currents (INa). P. minimum was before associated with neurotoxic symptoms, however, the nature and structure of toxins produced by this dinoflagellate remains unknown. Three P. minimum strains, ccmp1529, ccmp2811 and ccmp2956, growing in different conditions of temperature, salinity and light were used to study the production of toxic compounds. Electrophysiological assays showed no effect of ccmp2811 strain on INa, while ccmp1529 and ccmp2956 strains were able to significantly reduce INa in the same way as TTX. In these samples two new compounds, m/z 265 and m/z 308, were identified and characterized by liquid chromatography tandem high-resolution mass spectrometry. Besides, two TTX-related bacteria, Roseobacter and Vibrio sp, were observed. These results show for the first time that P. minimum produce TTX-like compounds with a similar ion pattern and C9-base to TTX analogues and with the same effect on INa.
Assuntos
Dinoflagellida/química , Contaminação de Alimentos , Inocuidade dos Alimentos , Alimentos Marinhos/análise , Frutos do Mar , Tetrodotoxina/análise , Animais , Bivalves/química , Dinoflagellida/crescimento & desenvolvimento , Análise de Perigos e Pontos Críticos de Controle , Espectrometria de MassasRESUMO
The marine environment constitutes an extraordinary resource for the discovery of new therapeutic agents. In the present manuscript we studied the effect of 3 different sponge derived guanidine alkaloids, crambescidine-816, -830, and -800. We show that these compounds strongly inhibit tumor cell proliferation by down-regulating cyclin-dependent kinases 2/6 and cyclins D/A expression while up-regulating the cell cyclin-dependent kinase inhibitors -2A, -2D and -1A. We also show that these guanidine compounds disrupt tumor cell adhesion and cytoskeletal integrity promoting the activation of the intrinsic apoptotic signaling, resulting in loss of mitochondrial membrane potential and concomitant caspase-3 cleavage and activation. The crambescidin 816 anti-tumor effect was fnally assayed in a zebrafish xenotransplantation model confirming its potent antitumor activity against colorectal carcinoma in vivo.Considering these results crambescidins could represent promising natural anticancer agents and therapeutic tools.
Assuntos
Alcaloides/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Guanidina/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Compostos de Espiro/farmacologia , Animais , Caspase 3/metabolismo , Adesão Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Relação Dose-Resposta a Droga , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Guanidina/farmacologia , Células HT29 , Células Hep G2 , Humanos , Concentração Inibidora 50 , Células MCF-7 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
The turbot is a flatfish (Pleuronectiformes) with increasing commercial value, which has prompted active genomic research aimed at more efficient selection. Here we present the sequence and annotation of the turbot genome, which represents a milestone for both boosting breeding programmes and ascertaining the origin and diversification of flatfish. We compare the turbot genome with model fish genomes to investigate teleost chromosome evolution. We observe a conserved macrosyntenic pattern within Percomorpha and identify large syntenic blocks within the turbot genome related to the teleost genome duplication. We identify gene family expansions and positive selection of genes associated with vision and metabolism of membrane lipids, which suggests adaptation to demersal lifestyle and to cold temperatures, respectively. Our data indicate a quick evolution and diversification of flatfish to adapt to benthic life and provide clues for understanding their controversial origin. Moreover, we investigate the genomic architecture of growth, sex determination and disease resistance, key traits for understanding local adaptation and boosting turbot production, by mapping candidate genes and previously reported quantitative trait loci. The genomic architecture of these productive traits has allowed the identification of candidate genes and enriched pathways that may represent useful information for future marker-assisted selection in turbot.
Assuntos
Adaptação Fisiológica , Linguados/genética , Genoma , Animais , Evolução Molecular , Proteínas de Peixes/genética , Anotação de Sequência Molecular , Fases de Leitura Aberta , Sequências Repetitivas de Ácido NucleicoRESUMO
Harmful algal blooms caused by phytoplankton can occur in all aquatic environments. Some of the algae present in these blooms are capable of producing extremely potent toxins. Due to climate change and eutrophication, harmful algal blooms are increasing on a global scale. One kind of toxin producing algae are those that produce okadaic acid, its derivatives (dinophysistoxin-1 and 2), and microcystins. These toxins are potent inhibitors of protein phosphatase 2A, so this protein is used to detect the mentioned toxins in natural samples. Originally protein phosphatase 2A purified from animal tissues was used, but enzyme activity and stability fluctuations prevented the use of the enzyme in detection kits. Expression of the enzyme as a recombinant protein provided a solution to this problem. For this purpose, several strategies have been followed. We evaluated the activity, specificity and stability of the human protein phosphatase 2A catalytic subunit α expressed in insect larvae and showed that this expression system can be a reliable source of high quantities of stable enzyme.