Avaliação da atividade citotóxica e antitumoral de acetohidroxamatos sintéticos em modelos in vitro e in vivo

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Farmacologia, Florianópolis, 2017.O tratamento do câncer ainda se apresenta como um grande desafio, principalmente devido à resistência das células tumorais aos quimioterápicos e à alta toxicidade dos tratamentos convencionais. Estes fatores têm impulsionado a pesquisa de novas estratégias terapêuticas na área da oncologia, focando principalmente em novas classes de medicamentos anticâncer. Alguns compostos que têm despertado interesse na área oncológica são os derivados de ácidos hidroxâmicos, uma vez que estes compostos têm apresentado efeito citotóxico e antitumoral, principalmente por meio de inibição de histona deacetilases. Neste trabalho foi investigado o efeito citotóxico in vitro de onze acetohidroxamatos, além do perfil farmacocinético e atividade antitumoral in vivo dos compostos mais promissores. Todos os compostos apresentaram atividade citotóxica frente às linhagens tumorais de melanoma (A375) e glioblastoma humano (U-87 MG) no ensaio de MTT, sendo que os compostos AKS 7, AKS 26 e AKS 61 destacaram-se pela alta potência e/ou pelas características físico-químicas promissoras, prosseguindo por esta razão para as próximas etapas. Destes, o composto AKS 61 foi o que apresentou capacidade antiproliferativa em concentrações mais baixas nos ensaios de Sulforrodamina B e de Clonogenicidade, além de apresentar menor citotoxicidade frente à linhagem não-tumoral de cardiomiócitos derivados de células-tronco pluripotentes induzidas (CMCiPS), tendo sido, portanto, selecionado para realizar alguns estudos mecanísticos in vitro. O AKS 61 bloqueou o ciclo celular de células da linhagem A375 de maneira bifásica (inicialmente em G2/M e posteriormente em G1), além de reduzir o potencial de membrana mitocondrial e induzir apoptose das células tratadas. Esse composto foi também eficaz em induzir autofagia após 24 horas de tratamento. A fim de avaliar o potencial antitumoral do AKS 61 in vivo, camundongos nude foram submetidos ao modelo de tumor xenográfico subcutâneo a partir da inoculação de células A375 e, em seguida, foram tratados com o composto AKS 61 pela via intravenosa (i.v.). O tratamento com AKS 61 (2 mg/kg, i.v.) não foi capaz de inibir o crescimento tumoral, provavelmente devido ao seu perfil farmacocinético desfavorável. Este composto apresentou tempo de meia vida plasmática inferior a quarenta minutos, acompanhada de uma alta taxa de eliminação total. Juntamente com a análise farmacocinética do AKS 61, foram também analisados os perfis dos compostos AKS 26 e AKS 7, sendo que este último apresentou o perfil mais favorável, inclusive apresentando biodisponibilidade pela via oral. Os resultados obtidos neste trabalho evidenciam o potencial desta série de compostos derivados do ácido hidroxâmico e guiarão a otimização dessas moléculas, com o objetivo de obter um composto eficaz in vivo que apresente características farmacocinéticas mais favoráveis para a continuidade do desenvolvimento não-clínico.Abstract : Cancer treatment is still challenging, mainly due to tumor resistance to chemotherapy and high toxicity of conventional therapy. These factors have driven the search of new therapy strategies and the discovery of better anticancer drugs. Some compounds that have aroused attention in oncology include the hydroxamic acid derivatives, once they have demonstrated cytotoxic and antitumor effect, mostly because of their histone deacetylases inhibition property. In the present study we have investigated the in vitro cytotoxic effect of eleven synthetic acetohydroxamates, as well as the in vivo pharmacokinetic profile and antitumor activity of most promising compounds. All compounds presented cytotoxic activity against A375 (human melanoma) and U-87 MG (human glioblastoma) cell lines in the MTT assay, and the compounds AKS 7, AKS 26 and AKS 61 stood out for their promising potency and/or physicochemical characteristics, therefore proceeding to the next steps. Of these, the compound AKS 61 was the one that presented the most favorable antiproliferative capacity in the Sulforhodamine B and Clonogenic assays, also presenting smaller cytotoxicity against the non-tumoral lineage CMCiPS (cardiomyocytes derived from human induced pluripotent stem cells), being therefore chosen to perform some mechanistic studies. AKS 61 was able to arrest the cell cycle of A375 cells biphasically (initially in G2/M and later in G1), in addition to inducing a decrease in the mitochondrial membrane potential and increasing apoptosis in treated cells. The compound was also capable of inducing autophagy after 24 hours of treatment. In order to assess the in vivo antitumor effect of AKS 61, nude mice were submitted to the xenographic tumor model (by A375 cells inoculation) and treated with the compound intravenously. AKS 61 treatment (2 mg/kg, i.v.) failed to inhibit tumor growth, probably because of its unfavorable pharmacokinetic profile. This compound presented a half-life time of less than 40 minutes, accompanied by a high clearance rate. Together with the pharmacokinetic analysis of AKS 61, the profiles of the compounds AKS 26 and AKS 7 were also analyzed, the latter having the most favorable profile and presenting oral bioavailability. These data show the potential anticancer effect of these hydroxamic acid derivatives and will guide the optimization of these molecules in order to obtain a more potent and effective in vivo compound associated with favorable pharmacokinetic characteristics to continue the nonclinical development

Cinnamomum zeylanicum Essential Oil Reduces Infestation by Alphitobius diaperinus in Poultry Litter

Background: Even though insecticides are managed and the period of sanitary emptiness in poultry is respected, the elimination of Alphitobius diaperinus may not be successful. The use of essential oils of plant origin presents as a good alternative in the substitution of insecticides with synthetic molecules, since they are easy to obtain, with rapid degradationand without risk of residues for non-target organisms. The main objective of the present study was to examine whether Cinnamomum zeylanicum oil reduces Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) infestations under experimental conditions, without causing toxicity to broilers chicks exposed to treated litter.Materials, Methods & Results: The experimental design was completely randomized, with four replications per treatment. The treatments were as follows: solvent control using the diluent Dimethyl Sulfoxide 5% (oil diluent); chemical control using 5 g/m² cypermethrin; one spray of C. zeylanicum 5% oil; and two sprays of C. zeylanicum 5% oil. Each experimental unit was infested with 150 lesser mealworm adults. At 15 days of the broiler chick’s life, blood was collected for biochemical analysis (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, globulin, triglycerides and uric acid), and liver fragments were isolated for histopathological analysis. Using TupeTrap devices, we counted lesser mealworm 40 days after treatment. The treatments did not alter biochemical variables, and did not causehistopathological lesions in liver. The treatments with C. zeylanicum 5% oil with one and two sprays efficiently reduced lesser mealworm infestation compared with solvent control. Cypermethrin treatment had no effect.Discussion: Many of the commercial products present low effectiveness in the control of A. diaperinus, since the target organisms develop resistance to the product. In the present study, we used a higher cypermethrin dose than that recommended by the manufacturer, in order to increase efficacy in the face of possible resistance. Even so, cypermethrin did not efficiently control the organism. The effectiveness of the essential oil of C. zeylanicum tested can be attributed to the compounds found in greater quantity in the oil composition, such as cinnamaldehyde (41.27%), linalool (13.05%) and methyl eugenol (10.87%), characterized as responsible for the action of oil repellency. Monoterpenoid compounds found in essential oils extracted from plants have insecticidal action acting on the central nervous system of insects, which impairs their development, being characterized as neurotoxic compounds. The results found with the essential oil of C. zeylanicum are of great importance, since the control of A. diaperinus is not efficient because this organism has behavior that favors reinfestation in the poultry houses, such as shelter in cracks, in the draperies, below the feeders and in the soil. The biochemical analyzes of the blood can as important tools to assist in the monitoring of broilers health, in the diagnosis and treatment of diseases, and therefore the results presented are of great importance since they assist in the search for alternative methods for the control of A. diaperinus, where we can affirm that the essential oil of C. zeylanicum does not cause toxicity to broilers. Based on these results we can affirm that essential oil of Cinnamomum zeylanicum, 5%, is an effective substitute for existing commercially-available insecticides.Keywords: alternative control, cinnamon oil, insecticide, lesser mealworm

From MYCN to inflammaging : understanding the pathogenesis of immature T-ALL

Although our understanding of the genetic basis of T-cell acute lymphoblastic leukemia (T-ALL) has improved with next-generation sequencing efforts, features underlying poor outcome remain unclear. To deconvolute the individual contributions of T-ALL oncogenes and generate more relevant experimental models, a recently developed, efficient and reproducible synthetic T-ALL model from primary human CD34+ cord blood cells was used. RNA-seq of synthetic leukemias showed that the same set of genetic alterations can lead to two distinct types of leukemia – one resembling uncommitted T-cell progenitors (Early) and another resembling committed, double positive T cells (Late). Early leukemias exhibited high MYCN expression, which correlated with poor clinical outcome in a large pediatric cohort. Knockout and knockdown experiments revealed Early leukemias to be dependent on MYCN for initial transformation and subsequent clonal propagation. Use of histone ChIP-Seq data revealed broad domains of H3K4 trimethylation extending across the entirety of the MYCN gene body. Knockdown of the lysine methyltransferase KMT2D via shRNA resulted in reduced expression of MYCN and severely restricted growth of Early leukemia cells. These findings suggest a prominent role for epigenetic regulation in maintaining consistent oncogene expression and defining the malignant identity of a particularly aggressive subtype of human leukemia. The fact that the same set of genetic alterations can lead to distinct leukemia subtypes suggested other variables such as cell developmental stage and microenvironment could influence the transformation process. Because adult patients present a worse prognosis in T-ALL and aging is accompanied by a progressive increase in the body’s proinflammatory microenvironment, I then employed our synthetic model to study how chronic inflammation can alter T-cell development and leukemogenesis. I found that chronic inflammatory stimulation delays T-cell differentiation and alters the growth and differentiation trajectory of transformed cells, which ultimately favors selection of more immature T-ALL subtypes. Overall, these studies identified genetic, epigenetic, and environmental features which contribute to the pathogenesis of immature T-ALLs. These results emphasize developmental stage-specific oncogene and cytokine dependencies which could inform the development of new therapeutic options.Medicine, Faculty ofGraduat

Tcf1 is essential for initiation of oncogenic Notch1-driven chromatin topology in T-ALL

Altres ajuts: The Swiss National Science Foundation (SNF 31003A_1467198 and SNF 310030_188505); The Swiss Cancer League; The US National Institutes of Health NIH grants P30 CA013696 and R35 CA210065NOTCH1 is a well-established lineage specifier for T cells and among the most frequently mutated genes throughout all subclasses of T cell acute lymphoblastic leukemia (T-ALL). How oncogenic NOTCH1 signaling launches a leukemia-prone chromatin landscape during T-ALL initiation is unknown. Here we demonstrate an essential role for the high-mobility-group transcription factor Tcf1 in orchestrating chromatin accessibility and topology, allowing aberrant Notch1 signaling to convey its oncogenic function. Although essential, Tcf1 is not sufficient to initiate leukemia. The formation of a leukemia-prone epigenetic landscape at the distal Notch1-regulated Myc enhancer, which is fundamental to this disease, is Tcf1-dependent and occurs within the earliest progenitor stage even before cells adopt a T lymphocyte or leukemic fate. Moreover, we discovered a unique evolutionarily conserved Tcf1-regulated enhancer element in the distal Myc-enhancer, which is important for the transition of preleukemic cells to full-blown disease

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria

Mechanisms Underlying the Scratching Behavior Induced by the Activation of Proteinase-Activated Receptor-4 in Mice

A role for proteinase-activated receptor-4 (PAR-4) was recently suggested in itch sensation. Here, we investigated the mechanisms underlying the pruriceptive actions of the selective PAR-4 agonist AYPGKF-NH2 (AYP) in mice. Dorsal intradermal (i.d.) administration of AYP elicited intense scratching behavior in mice, which was prevented by the selective PAR-4 antagonist (pepducin P4pal-10). PAR-4 was found to be coexpressed in 32% of tryptase-positive skin mast cells, and AYP caused a 2-fold increase in mast cell degranulation. However, neither the treatment with cromolyn nor the deficiency of mast cells (WBB6F1-KitW/Wv mice) was able to affect AYP-induced itch. PAR-4 was also found on gastrin-releasing peptide (GRP)-positive neurons (pruriceptive fibers), and AYP-induced itch was reduced by the selective GRP receptor antagonist RC-3095. In addition, AYP evoked calcium influx in ∼1.5% of cultured DRG neurons also sensitive to TRPV1 (capsaicin) and/or TRPA1 (AITC) agonists. Importantly, AYP-induced itch was reduced by treatment with either the selective TRPV1 (SB366791), TRPA1 (HC-030031), or NK1 (FK888) receptor antagonists. However, genetic loss of TRPV1, but not of TRPA1, diminished AYP-induced calcium influx in DRG neurons and the scratching behavior in mice. These findings provide evidence that PAR-4 activation by AYP causes pruriceptive itch in mice via a TRPV1/TRPA1-dependent mechanism

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

Submitted by Nuzia Santos ([email protected]) on 2015-02-27T12:50:33Z No. of bitstreams: 1 2014_112.pdf: 385803 bytes, checksum: 73ae8e5baf37fce92c3d713b7f570aed (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2015-02-27T12:50:39Z (GMT) No. of bitstreams: 1 2014_112.pdf: 385803 bytes, checksum: 73ae8e5baf37fce92c3d713b7f570aed (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2015-02-27T12:56:22Z (GMT) No. of bitstreams: 1 2014_112.pdf: 385803 bytes, checksum: 73ae8e5baf37fce92c3d713b7f570aed (MD5)Made available in DSpace on 2015-02-27T12:56:22Z (GMT). No. of bitstreams: 1 2014_112.pdf: 385803 bytes, checksum: 73ae8e5baf37fce92c3d713b7f570aed (MD5) Previous issue date: 2014CNPq, LMW and MLBUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Protozoologia. Florianópolis, SC, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Imunologia Celular e Molecular. Belo Horizonte, MG, BrasilLaboratório Central do Estado de Santa Catarina. Florianópolis, SC, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Microbiologia. Laboratório de Vírus. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Microbiologia. Laboratório de Vírus. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Microbiologia. Laboratório de Vírus. Belo Horizonte, MG, BrasilThe identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteri