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1.
Chem Biol Interact ; 311: 108789, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31401089

RESUMO

The cytotoxicity of a dinuclear imine-copper (II) complex 2, and its analogous mononuclear complex 1, toward different melanoma cells, particularly human SKMEL-05 and SKMEL-147, was investigated. Complex 2, a tyrosinase mimic, showed much higher activity in comparison to complex 1, and its reactivity was verified to be remarkably activated by UVB-light, while the mononuclear compound showed a small or negligible effect. Further, a significant dependence on the melanin content in the tumor cells, both from intrinsic pigmentation or stimulated by irradiation, was observed in the case of complex 2. Similar tests with keratinocytes and melanocytes indicated a much lower sensitivity to both copper (II) complexes, even after exposition to UV light. Clonogenic assays attested that the fractions of melanoma cells survival were much lower under treatment with complex 2 compared to complex 1, both with or without previous irradiation of the cells. The process also involves generation of reactive oxygen species (ROS), as verified by EPR spectroscopy, and by using fluorescence indicators. Autophagic assays indicated a remarkable formation of cytoplasmic vacuoles in melanomas treated with complex 2, while this effect was not observed in similar treatment with complex 1. Monitoring of specific protein LC3 corroborated the simultaneous occurrence of autophagy. A balance interplay between different modes of cell death, apoptosis and autophagy, occurs when melanomas were treated with the dinuclear complex 2, in contrast to the mononuclear complex 1. These results pointed out to different mechanisms of action of such complexes, depending on its nuclearity.


Assuntos
Complexos de Coordenação/química , Cobre/química , Iminas/química , Monofenol Mono-Oxigenase/metabolismo , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Complexos de Coordenação/metabolismo , Complexos de Coordenação/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos da radiação , Humanos , Melaninas/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tubulina (Proteína)/metabolismo , Raios Ultravioleta
2.
Expert Opin Ther Pat ; 29(8): 623-641, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31353978

RESUMO

Introduction: About 20 patents have been published from 2013 to 2018 for developing advanced cancer therapeutics by targeting tubulin polymerization. Currently, there are several tubulin inhibitors that are in the drug development pipeline for various cancers alone or in combination including antibody-conjugated drugs (ACDs). Areas covered: Important patents focusing on the development of tubulin inhibitors published from 2013 to 2018 are covered. This review mainly focuses on the tubulin inhibitors that are being synthesized and studied in cancer research along with their structures and their phases of development in preclinical and clinical research. Expert opinion: Regulation of microtubules is important for cell division, cell motility, intracellular transport, and cell shape maintenance. Modulating its activity proved to be very effective in various diseases including different types of cancers. Microtubules are composed of two units, namely, alpha and beta-tubulin, and modifications at these ends affect both its functions and dynamics. A number of compounds that have been designed and synthesized bearing various heterocyclic scaffolds have been proven to modulate its activity and have emerged as potent tubulin inhibitors. This encourages more to study microtubules in order to find a variety of novel, potent compounds as anticancer drugs.


Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Moduladores de Tubulina/farmacologia , Animais , Antineoplásicos/química , Desenho de Drogas , Desenvolvimento de Medicamentos/métodos , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Neoplasias/patologia , Patentes como Assunto , Relação Estrutura-Atividade , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química
3.
Eur J Med Chem ; 178: 116-130, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31177073

RESUMO

In this study, a series of novel HDAC inhibitors, using 1,2,4-oxadiazole-containing as the cap group, were synthesized and evaluated in vitro. Compound 14b, N-hydroxy-2-(methyl((3-(1-(4-methylbenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methyl)amino)pyrimidine-5-carboxamide, displayed the most potent histone deacetylase (HDAC) inhibition, especially against HDAC1, 2, and 3 with IC50 values of 1.8, 3.6 and 3.0 nM, respectively. In vitro antiproliferative studies confirmed that 14b was more potent than SAHA, with IC50 values against 12 types of cancer cell lines ranging from 9.8 to 44.9 nM. The results of Western blot assays showed that compound 14b can significantly up-regulate the acetylation of the biomarker his-H3 and molecular docking analyses revealed the mode of action of compound 14b against HDAC1. The results of flow-cytometry analysis suggested that compound 14b induces cell cycle arrest at the G1 phase and has apoptotic effects. Further investigation of the activity of 14b on the primary cells of three patients, showed IC50 values of 21.3, 61.1, and 77.4 nM. More importantly, an oral bioavailability of up to 53.52% was observed for 14b. An in vivo pharmacodynamic evaluation demonstrated that compound 14b can significantly inhibit tumor growth in a Daudi Burkitt's lymphoma xenograft model, with tumor inhibition rates of 53.8 and 46.1% observed at 20 and 10 mg/kg when administered p.o. and i.v., respectively. These results indicate that compound 14b may be a suitable lead for further evaluation and development as an HDAC inhibitor and a potent anticancer agent.


Assuntos
Antineoplásicos/uso terapêutico , Linfoma de Burkitt/tratamento farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Oxidiazóis/uso terapêutico , Acetilação/efeitos dos fármacos , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Histona Desacetilase 1/química , Histona Desacetilase 1/metabolismo , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/farmacocinética , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/farmacocinética , Camundongos Endogâmicos NOD , Camundongos SCID , Simulação de Acoplamento Molecular , Estrutura Molecular , Oxidiazóis/síntese química , Oxidiazóis/farmacocinética , Ligação Proteica , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Eur J Med Chem ; 178: 177-194, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31185410

RESUMO

Microtubule is one of the important targets for cancer treatment. A novel class of diaryl substituted imidazo[4,5-c]pyridin-2-ones and imidazo[4,5-c]pyridines were designed based on combination principles by merging the structures of ß-lactams and purine-type compounds known as tubulin polymerization inhibitor and katanin activity up-regulator, respectively. Their antitumor activities were evaluated in vitro and the mechanism was elucidated, leading to the identification of 1,6-diaryl-1H-imidazo[4,5-c]pyridin-2(3H)-one 20b as the first bifunctional agent that can target both tubulin and katanin simultaneously. The in vivo assays verified that compound 20b significantly inhibited xenograft tumor growth with good pharmacokinetic characteristics, demonstrating a promising potential for further development into anti-tumor drug candidates with a unique mechanism of dual-targeting microtubule.


Assuntos
Antineoplásicos/uso terapêutico , Imidazóis/uso terapêutico , Katanina/antagonistas & inibidores , Piridinas/uso terapêutico , Moduladores de Tubulina/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacocinética , Apoptose/efeitos dos fármacos , Carcinoma Endometrioide/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Imidazóis/síntese química , Imidazóis/química , Imidazóis/farmacocinética , Katanina/metabolismo , Ligantes , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos Nus , Estrutura Molecular , Neoplasias Ovarianas/tratamento farmacológico , Piridinas/síntese química , Piridinas/química , Piridinas/farmacocinética , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacocinética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Eur J Med Chem ; 177: 448-456, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31174062

RESUMO

The colchicine site inhibitors (CSIs) showed promising prospects as antitumor agents due to their vascular disrupting activities besides antimitotic activities. 1-Phenyl-dihydrobenzoindazole was found as a novel scaffold of CSI without the cis-trans isomerization problem. The X-ray co-crystal structure of the lead compound with tubulin was determined, which revealed the binding mode including special water-bridged hydrogen bonds. The structure also provided guidance for the structural optimization of this type of CSI, which led to the discovery of the most potent inhibitor A3, with growth IC50 lower than 1 nM against human colon cancer cell lines and tubulin polymerization IC50 of 1.6 µM. In addition, its water-soluble prodrug B1 showed good in vivo antitumor activity on two human colon cancer xenograft nude mice models, encouraging further study of this type of antitumor compound.


Assuntos
Antineoplásicos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Indazóis/farmacologia , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Feminino , Células HCT116 , Humanos , Indazóis/síntese química , Indazóis/química , Indazóis/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Ligação Proteica/efeitos dos fármacos , Solubilidade , Tubulina (Proteína)/química , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Eur J Med Chem ; 178: 287-296, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31195170

RESUMO

Structure-activity relationships for rigid analogues of combretastatin A-4 (CA-4) were investigated, leading to the discovery of a series of 3,4-diaryl-1,2,5-oxadiazole-N-oxides. Among them, 7n' and 7n'' showed remarkable antiproliferative activities against three cancer cell lines in nanomolar concentrations. Interestingly, 7n' inhibited tubulin polymerization much more efficiently than CA-4. Cellular mechanism investigation elucidated 7n' disrupted the cellular microtubule structure, arrested cell cycle at G2/M phase and induces apoptosis. Molecular modeling study revealed 1,2,5-oxadiazole-N-oxide ring could increase a hydrogen bond interaction with the binding site. These results provide impetus and further guidance for the development of new CA-4 analogues.


Assuntos
Antineoplásicos/farmacologia , Desenho de Drogas , Oxidiazóis/farmacologia , Óxidos/farmacologia , Tubulina (Proteína)/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Estrutura Molecular , Oxidiazóis/síntese química , Oxidiazóis/química , Óxidos/síntese química , Óxidos/química , Polimerização/efeitos dos fármacos , Relação Estrutura-Atividade
7.
Cancer Sci ; 110(7): 2296-2308, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31074083

RESUMO

Vasohibin-2 (VASH2) is expressed in various cancers and promotes their progression. We recently reported that pancreatic cancer patients with higher VASH2 expression show poorer prognosis. Herein, we sought to characterize the role of VASH2 in pancreatic cancer. We used LSL-KrasG12D ; LSL-Trp53R172H ; Pdx-1-Cre (KPC) mice, a mouse model of pancreatic ductal adenocarcinoma (PDAC), and cells isolated from them (KPC cells). Knockdown of Vash2 from PDAC cells did not affect their proliferation, but decreased their migration. When Vash2-knockdown PDAC cells were orthotopically inoculated, liver metastasis and peritoneal dissemination were reduced, and the survival period was significantly prolonged. When KPC mice were crossed with Vash2-deficient mice, metastasis was significantly decreased in Vash2-deficient KPC mice. VASH2 was recently identified to have tubulin carboxypeptidase activity. VASH2 knockdown decreased, whereas VASH2 overexpression increased tubulin detyrosination of PDAC cells, and tubulin carboxypeptidase (TCP) inhibitor parthenolide inhibited VASH2-induced cell migration. We next clarified its role in the tumor microenvironment. Tumor angiogenesis was significantly abrogated in vivo when VASH2 was knocked down or deleted. We further examined genes downregulated by Vash2 knockdown in KPC cells, and found chemokines and cytokines that were responsible for the recruitment of myeloid derived suppressor cells (MDSC). Indeed, MDSC were accumulated in PDAC of KPC mice, and they were significantly decreased in Vash2-deficient KPC mice. These findings suggest that VASH2 plays an essential role in the metastasis of PDAC with multiple effects on both cancer cells and the tumor microenvironment, including tubulin detyrosination, tumor angiogenesis and evasion of tumor immunity.


Assuntos
Proteínas Angiogênicas/genética , Proteínas Angiogênicas/metabolismo , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Regulação para Cima , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Tubulina (Proteína)/metabolismo , Microambiente Tumoral
8.
Artigo em Inglês | MEDLINE | ID: mdl-31125928

RESUMO

The colchicine binding site of tubulin is often used to screen the anti-mitotic compounds, which are widely used as anti-cancer therapies. In the present work, an affinity probe capillary electrophoresis (APCE) method was developed for determining the affinity of anti-mitotic compounds. To this end, a fluorescently labeled affinity probe, 5-carboxyfluorescein-colchicine (F-colchicine), was prepared for the affinity competition experiment. The probe can form a stable complex with tubulin with the binding stoichiometry of 0.75, and the dissociation constant Kd of the complex was determined as 5.7 × 10-5 mol/L. In the affinity competition experiment, F-colchicine was incubated with tubulin and the test compound in the solution. The F-colchicine-tubulin complexes and free F-colchicine were quickly separated by CE and the concentration of free F-colchicine was accurately determined with the laser induced fluorescence detection. The affinity constant of the tested compound can be measured with the affinity competition binding curve. The enantiomers of the anti-mitotic compound were evaluated by using the method. The binding affinity of the enantiomers displayed an enantioselective manner. Compared to other affinity binding assay methods, our method is more straightforward, more accurate, and more cost-effective.


Assuntos
Antimitóticos , Colchicina/metabolismo , Descoberta de Drogas/métodos , Eletroforese Capilar/métodos , Tubulina (Proteína)/metabolismo , Antimitóticos/análise , Antimitóticos/química , Antimitóticos/metabolismo , Sítios de Ligação , Colchicina/química , Fluoresceínas/química , Corantes Fluorescentes/química , Ligação Proteica , Reprodutibilidade dos Testes , Tubulina (Proteína)/química
9.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052191

RESUMO

Tubulins and microtubules (MTs) represent targets for taxane-based chemotherapy. To date, several lines of evidence suggest that effectiveness of compounds binding tubulin often relies on different post-translational modifications on tubulins. Among them, methylation was recently associated to drug resistance mechanisms impairing taxanes binding. The sea urchin is recognized as a research model in several fields including fertilization, embryo development and toxicology. To date, some α- and ß-tubulin genes have been identified in P. lividus, while no data are available in echinoderms for arginine methyl transferases (PRMT). To evaluate the exploiting of the sea urchin embryo in the field of antiproliferative drug development, we carried out a survey of the expressed α- and ß-tubulin gene sets, together with a comprehensive analysis of the PRMT gene family and of the methylable arginine residues in P. lividus tubulins. Because of their specificities, the sea urchin embryo may represent an interesting tool for dissecting mechanisms of tubulin targeting drug action. Therefore, results herein reported provide evidences supporting the P. lividus embryo as animal system for testing antiproliferative drugs.


Assuntos
Citostáticos/toxicidade , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Ouriços-do-Mar/efeitos dos fármacos , Testes de Toxicidade/métodos , Moduladores de Tubulina/toxicidade , Tubulina (Proteína)/metabolismo , Animais , Embrião não Mamífero/efeitos dos fármacos , Metilação , Processamento de Proteína Pós-Traducional , Ouriços-do-Mar/embriologia
10.
Cell Mol Life Sci ; 76(18): 3621-3640, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30953095

RESUMO

α-Tubulin acetyltransferase 1 (ATAT1) catalyzes acetylation of α-tubulin at lysine 40 in various organisms ranging from Tetrahymena to humans. Despite the importance in mammals suggested by studies of cultured cells, the mouse Atat1 gene is non-essential for survival, raising an intriguing question about its real functions in vivo. To address this question, we systematically analyzed a mouse strain lacking the gene. The analyses revealed that starting at postnatal day 5, the mutant mice display enlarged lateral ventricles in the forebrain, resembling ventricular dilation in human patients with ventriculomegaly. In the mice, ventricular dilation is due to hypoplasia in the septum and striatum. Behavioral tests of the mice uncovered deficits in motor coordination. Birth-dating experiments revealed that neuronal migration to the mutant septum and striatum is impaired during brain development. In the mutant embryonic fibroblasts, we found mild defects in cell proliferation and primary cilium formation. Notably, in these cells, ATAT1 is indispensable for tubulin hyperacetylation in response to high salt, high glucose, and hydrogen peroxide-induced oxidative stress. We investigated the role of ATAT1 in the hematopoietic system using multicolor flow cytometry and found that this system remains normal in the mutant mice. Although tubulin acetylation was undetectable in a majority of mutant tissues, residual levels were detected in the heart, skeletal muscle, trachea, oviduct, thymus and spleen. This study thus not only establishes the importance of ATAT1 in regulating mouse forebrain development and governing tubulin hyperacetylation during stress responses, but also suggests the existence of an additional α-tubulin acetyltransferase.


Assuntos
Acetiltransferases/metabolismo , Proteínas dos Microtúbulos/metabolismo , Estresse Oxidativo , Prosencéfalo/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação/efeitos dos fármacos , Acetiltransferases/genética , Animais , Comportamento Animal , Movimento Celular , Proliferação de Células , Células Cultivadas , Cílios/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microtúbulos/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , Estresse Oxidativo/efeitos dos fármacos , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/patologia
11.
Gynecol Oncol ; 154(1): 228-235, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31003747

RESUMO

OBJECTIVE: Recently, our laboratory identified sensory innervation within head and neck squamous cell carcinomas (HNSCCs) and subsequently defined a mechanism whereby HNSCCs promote their own innervation via the release of exosomes that stimulate neurite outgrowth. Interestingly, we noted that exosomes from human papillomavirus (HPV)-positive cell lines were more effective at promoting neurite outgrowth than those from HPV-negative cell lines. As nearly all cervical tumors are HPV-positive, we hypothesized that these findings would extend to cervical cancer. METHODS: We use an in vitro assay with PC12 cells to quantify the axonogenic potential of cervical cancer exosomes. PC12 cells are treated with cancer-derived exosomes, stained with the pan-neuronal marker (ß-III tubulin) and the number of neurites quantified. To assess innervation in cervical cancer, we immunohistochemically stained cervical cancer patient samples for ß-III tubulin and TRPV1 (sensory marker) and compared the staining to normal cervix. RESULTS: Here, we show the presence of sensory nerves within human cervical tumors. Additionally, we show that exosomes derived from HPV-positive cervical cancer cell lines effectively stimulate neurite outgrowth. CONCLUSIONS: These data identify sensory nerves as components of the cervical cancer microenvironment and suggest that tumor- derived exosomes promote their recruitment.


Assuntos
Vias Aferentes/patologia , Exossomos/patologia , Neoplasias do Colo do Útero/patologia , Vias Aferentes/metabolismo , Animais , Colo do Útero/inervação , Exossomos/metabolismo , Feminino , Células HeLa , Papillomavirus Humano 16/isolamento & purificação , Humanos , Imuno-Histoquímica , Neuritos/metabolismo , Neuritos/patologia , Células PC12 , Ratos , Canais de Cátion TRPV/metabolismo , Tubulina (Proteína)/metabolismo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologia
12.
Phytomedicine ; 58: 152770, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31005716

RESUMO

BACKGROUND: Phenanthrenes isolated from Juncus species possess different biological activities, including antiproliferative and antimigratory effects. PURPOSE: In this study, nine phenanthrenes isolated from the roots of Juncus inflexus were investigated for their antiproliferative activity on several gynecological cancer cell lines, using non-cancerous cells as controls. METHODS: Antiproliferative activities of the compounds were determined by means of MTT assay. Flow cytometry was used for cell cycle analysis and determination of mitotic cells. Activities of caspase-3, -8, and -9 were detected by colorimetric kits. Tubulin polymerization was followed by kinetic absorbance determination. Action on tumor cell migration was described using wound healing assay. Western blot assays were used to determine apoptosis-related factors at protein level. RESULTS: Among the compounds tested, juncusol exhibited the most substantial antiproliferative effect against cervical cancer HeLa cells. It was also revealed that juncusol has a distinct growth inhibitory effect in cervical cancer cell lines of various HPV status: it was highly active in HPV type 18-positive HeLa cells, while it was inactive in HPV type 16-positive SiHa and CaSki cells. Cell cycle analysis showed an increase in G2/M and subG1 cell populations after juncusol treatment. Caspase-3, -8, and -9 were detected to be activated by juncusol in HeLa cells, indicating that juncusol induces apoptotic cell death. Moreover, juncusol inhibited tubulin polymerization, as well as EGFR activation, suggesting two possible additional mechanisms that may account for juncusol's inducing a G2/M-phase cell cycle arrest and inhibiting cell migration. CONCLUSION: These results suggest that juncusol is a potent antiproliferative agent against HPV-18 related cervical cancer and may be considered as a lead compound for the development of innovative anticancer agents.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Magnoliopsida/química , Fenantrenos/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Antineoplásicos/química , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Humanos , Fenantrenos/química , Multimerização Proteica/efeitos dos fármacos , Tubulina (Proteína)/metabolismo
13.
Chem Pharm Bull (Tokyo) ; 67(7): 725-728, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30982797

RESUMO

Eighteen novel chalcone derivatives containing indole and naphthalene moieties (2-19) were synthesized and characterized by 1H-NMR, 13C-NMR and high resolution (HR)-MS spectra. All compounds were evaluated for their in vitro cytotoxic potential against human hepatocellular carcinoma (HepG2), human colon carcinoma (HCT116) and human breast adenocarcinoma (MCF-7) cell lines. Among them, compound 2, 3, 4 and 7 showed potent activities against tested cancer cell lines. More significantly, compound 7 exhibited the most potent cytotoxic activity against HepG2, HCT116 and MCF-7 with IC50 values of 0.65, 1.13 and 0.82 µM, respectively. Furthermore, flow cytometry analysis indicated that compound 7 arrested cancer cells in G2/M phase. The compound 7 also displayed significant inhibition of tubulin polymerization (IC50 = 3.9 µM). Finally, molecular docking studies were performed to explore the possible interactions between compound 7 and tubulin binding pockets.


Assuntos
Antineoplásicos/síntese química , Chalconas/química , Indóis/química , Naftalenos/química , Moduladores de Tubulina/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Chalconas/metabolismo , Chalconas/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacologia
14.
Molecules ; 24(7)2019 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-30935100

RESUMO

Monoterpenoid indole alkaloids are structurally diverse natural products found in plants of the family Apocynaceae. Among them, vincristine and its derivatives are well known for their anticancer activity. Bousigonia mekongensis, a species in this family, contains various monoterpenoid indole alkaloids. In the current study, fourteen known aspidosperma-type monoterpenoid indole alkaloids (1⁻14) were isolated and identified from a methanol extract of the twigs and leaves of B. mekongensis for the first time. Among them, compounds 3, 6, 9, and 13 exhibited similar antiproliferative activity spectra against A549, KB, and multidrug-resistant (MDR) KB subline KB-VIN cells with IC50 values ranging from 0.5⁻0.9 µM. The above alkaloids efficiently induced cell cycle arrest at the G2/M phase by inhibiting tubulin polymerization as well as mitotic bipolar spindle formation. Computer modeling studies indicated that compound 7 likely forms a hydrogen bond (H-bond) with α- or ß-tubulin at the colchicine site. Evaluation of the antiproliferative effects and SAR analysis suggested that a 14,15-double bond or 3α-acetonyl group is critical for enhanced antiproliferative activity. Mechanism of action studies demonstrated for the first time that compounds 3, 4, 6, 7, and 13 efficiently induce cell cycle arrest at G2/M by inhibiting tubulin polymerization by binding to the colchicine site.


Assuntos
Aspidosperma/química , Multimerização Proteica/efeitos dos fármacos , Alcaloides de Triptamina e Secologanina/química , Alcaloides de Triptamina e Secologanina/farmacologia , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo
15.
Parasitol Res ; 118(6): 1899-1918, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30949853

RESUMO

After host cell invasion, Toxoplasma secretes a variety of dense granule proteins (GRA proteins) from its secretory dense granules, which are involved in the biogenesis of the parasitophorous vacuole (PV). TgGRA8I is predicted to contain proline-rich domains, which are structural features of some cytoskeleton-related proteins. In agreement with this observation, previous proteomic analyses revealed the presence of TgGRA8I in the Toxoplasma sub-pellicular cytoskeleton. In the present study, we show (1) by docking analyses that TgGRA8I may interact with both Toxoplasma ß-tubulin and actin; (2) by immunoelectron microscopy, proteomic, biochemical, and cellular approaches that TgGRA8I associates with sub-pellicular microtubules and actin at the parasite sub-pellicular cytoskeleton; (3) that type I parasites (RH strain) lacking the GRA8 gene (RHΔku80Δgra8) exhibit loss of conoid extrusion, diminished cell infection, and egress capabilities, and that these motility impairments were likely due to important alterations in their sub-pellicular cytoskeleton, in particular their sub-pellicular microtubules and meshwork. Parasites lacking the GRA4 gene (RHΔku80Δgra4) did not show modifications in the organization of the sub-pellicular cytoskeleton. Collectively, these results demonstrated that TgGRA8I is a dense granule protein that, besides its role in the formation of the PV, contributes to the organization of the parasite sub-pellicular cytoskeleton and motility. This is the first proline-rich protein described in the Toxoplasma cytoskeleton, which is a key organelle for both the parasite motility and the invasion process. Knowledge about the function of cytoskeleton components in Toxoplasma is fundamental to understand the motility process and the host cell invasion mechanism. Refining this knowledge should lead to the design of novel pharmacological strategies for the treatment against toxoplasmosis.


Assuntos
Actinas/metabolismo , Antígenos de Protozoários/metabolismo , Movimento Celular/genética , Citoesqueleto/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Toxoplasma/patogenicidade , Tubulina (Proteína)/metabolismo , Animais , Antígenos de Protozoários/genética , Transporte Biológico , Microscopia Imunoeletrônica , Microtúbulos/metabolismo , Simulação de Acoplamento Molecular , Proteômica , Proteínas de Protozoários/genética , Vesículas Secretórias/metabolismo , Toxoplasma/genética , Toxoplasmose/parasitologia , Toxoplasmose/patologia , Vacúolos/parasitologia
16.
BMC Evol Biol ; 19(1): 86, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30961520

RESUMO

BACKGROUND: The Scalidophora (Kinorhyncha, Loricifera and Priapulida) have an important phylogenetic position as early branching ecdysozoans, yet the architecture of their nervous organ systems is notably underinvestigated. Without such information, and in the absence of a stable phylogenetic context, we are inhibited from producing adequate hypotheses about the evolution and diversification of ecdysozoan nervous systems. Here, we utilize confocal laser scanning microscopy to characterize serotonergic, tubulinergic and FMRFamidergic immunoreactivity patterns in a comparative neuroanatomical study with three species of Echinoderes, the most speciose, abundant and diverse genus within Kinorhyncha. RESULTS: Neuroanatomy in Echinoderes as revealed by acetylated α-tubulin immunoreactivity includes a circumpharyngeal brain and ten neurite bundles in the head region that converge into five longitudinal nerves within the trunk. The ventral nerve cord is ganglionated, emerging from the brain with two connectives that converge in trunk segments 2-3, and diverge again within segment 8. The longitudinal nerves and ventral nerve cord are connected by two transverse neurites in segments 2-9. Differences among species correlate with the number, position and innervation of cuticular structures along the body. Patterns of serotoninergic and FMRFamidergic immunoreactivity correlate with the position of the brain neuropil and the ventral nerve cord. Distinct serotonergic and FMRFamidergic somata are associated with the brain neuropil and specific trunk segments along the ventral nerve cord. CONCLUSIONS: Neural architecture is highly conserved across all three species, suggesting that our results reveal a pattern that is common to more than 40% of the species within Kinorhyncha. The nervous system of Echinoderes is segmented along most of the trunk; however, posterior trunk segments exhibit modifications that are likely associated with sensorial, motor or reproductive functions. Although all kinorhynchs show some evidence of an externally segmented trunk, it is unclear whether external segmentation matches internal segmentation of nervous and muscular organ systems across Kinorhyncha, as we observed in Echinoderes. The neuroanatomical data provided in this study not only expand the limited knowledge on kinorhynch nervous systems but also establish a comparative morphological framework within Scalidophora that will support broader inferences about the evolution of neural architecture among the deepest branching lineages of the Ecdysozoa.


Assuntos
Eucariotos/fisiologia , Microscopia Confocal/métodos , Sistema Nervoso/anatomia & histologia , Neuroanatomia , Acetilação , Animais , FMRFamida/metabolismo , Filogenia , Serotonina/metabolismo , Tubulina (Proteína)/metabolismo
17.
Eur J Med Chem ; 171: 310-331, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30953881

RESUMO

Microtubules are a protein which is made of α- and ß-heterodimer. It is one of the main components of the cell which play a vital role in cell division especially in G2/M-phase. It exists in equilibrium dynamic of polymerization and depolymerization of α- and ß-heterodimer. It is one of the best targets for developing anti-cancer drugs. Various natural occurring molecules are well known for their anti-tubulin effect such as vinca, paclitaxel, combretastatin, colchicine etc. These microtubule-targeted drugs are acted through two processes (i) inhibiting depolymerization of tubulin (tubulin stabilizing agents) and (ii) inhibiting polymerization of tubulin (tubulin destabilizing agents). Now days, various binding domains have been explore through which these molecules are binding to tubulin but the three major binding domain of tubulin are taxol, vinca and colchicine binding domain. The present article mainly focus on the classification of various naturally occurring compounds on the basis of their inhibition processes (depolymerization and polymerization) and the site of interaction (targets taxol, vinca and colchicine binding domain) which has been hitherto reported. By placing all the naturally occurring taxol, vinca and colchicine binding site analogues at one place makes a better understanding of the tubulin interactions with known natural tubulin binders that would helps in the discovery of new and potent natural, semi-synthetic and synthetic analogues for treating cancer.


Assuntos
Produtos Biológicos/farmacologia , Colchicina/farmacologia , Paclitaxel/farmacologia , Tubulina (Proteína)/metabolismo , Vinca/química , Sítios de Ligação/efeitos dos fármacos , Produtos Biológicos/síntese química , Produtos Biológicos/química , Colchicina/síntese química , Colchicina/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Paclitaxel/síntese química , Paclitaxel/química , Polimerização/efeitos dos fármacos , Relação Estrutura-Atividade
18.
Cell Mol Life Sci ; 76(11): 2217-2229, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30980108

RESUMO

As the female gamete, meiotic oocytes provide not only half of the genome but also almost all stores for fertilization and early embryonic development. Because de novo mRNA transcription is absent in oocyte meiosis, protein-level regulations, especially the ubiquitin proteasome system, are more crucial. As the largest family of ubiquitin E3 ligases, Skp1-Cullin-F-box complexes recognize their substrates via F-box proteins with substrate-selected specificity. However, the variety of F-box proteins and their unknown substrates hinder our understanding of their functions. In this report, we find that Fbxo30, a new member of F-box proteins, is enriched in mouse oocytes, and its expression level declines substantially after the metaphase of the first meiosis (MI). Notably, depletion of Fbxo30 causes significant chromosome compaction accompanied by chromosome segregation failure and arrest at the MI stage, and this arrest is not caused by over-activation of spindle assembly checkpoint. Using immunoprecipitation and mass spectrometric analysis, we identify stem-loop-binding protein (SLBP) as a novel substrate of Fbxo30. SLBP overexpression caused by Fbxo30 depletion results in a remarkable overload of histone H3 on chromosomes that excessively condenses chromosomes and inhibits chromosome segregation. Our finding uncovers an unidentified pathway-controlling chromosome segregation and cell progress.


Assuntos
Segregação de Cromossomos , Cromossomos de Mamíferos/metabolismo , Proteínas F-Box/genética , Histonas/genética , Meiose , Proteínas Nucleares/genética , Oócitos/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Animais , Cromossomos de Mamíferos/ultraestrutura , Proteínas F-Box/antagonistas & inibidores , Proteínas F-Box/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Proteínas Nucleares/metabolismo , Oócitos/ultraestrutura , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
19.
Nat Commun ; 10(1): 1838, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015426

RESUMO

Cilia and flagella play essential roles in cell motility, sensing and development. These organelles have tightly controlled lengths, and the axoneme, which forms the core structure, has exceptionally high stability. This is despite being composed of microtubules that are often characterized as highly dynamic. To understand how ciliary tubulin contribute to stability, we develop a procedure to differentially extract tubulins from different components of axonemes purified from Chlamydomonas reinhardtii, and characterize their properties. We find that the microtubules support length stability by two distinct mechanisms: low dynamicity, and unusual stability of the protofilaments. The high stability of the protofilaments manifests itself in the formation of curved tip structures, up to a few microns long. These structures likely reflect intrinsic curvature of GTP or GDP·Pi tubulin and provide structural insights into the GTP-cap. Together, our study provides insights into growth, stability and the role of post-translational modifications of axonemal microtubules.


Assuntos
Axonema/metabolismo , Movimento Celular , Cílios/fisiologia , Proteínas de Plantas/metabolismo , Tubulina (Proteína)/metabolismo , Chlamydomonas reinhardtii , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Plantas/isolamento & purificação , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/isolamento & purificação
20.
Nat Protoc ; 14(5): 1634-1660, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30996262

RESUMO

In vitro reconstitutions of microtubule assemblies have provided essential mechanistic insights into the molecular bases of microtubule dynamics and their interactions with associated proteins. The tubulin code has emerged as a regulatory mechanism for microtubule functions, which suggests that tubulin isotypes and post-translational modifications (PTMs) play important roles in controlling microtubule functions. To investigate the tubulin code mechanism, it is essential to analyze different tubulin variants in vitro. Until now, this has been difficult, as most reconstitution experiments have used heavily post-translationally modified tubulin purified from brain tissue. Therefore, we developed a protocol that allows purification of tubulin with controlled PTMs from limited sources through cycles of polymerization and depolymerization. Although alternative protocols using affinity purification of tubulin also yield very pure tubulin, our protocol has the unique advantage of selecting for fully functional tubulin, as non-polymerizable tubulin is excluded in the successive polymerization cycles. It thus provides a novel procedure for obtaining tubulin with controlled PTMs for in vitro reconstitution experiments. We describe specific procedures for tubulin purification from adherent cells, cells grown in suspension cultures and single mouse brains. The protocol can be combined with drug treatment, transfection of cells before tubulin purification or enzymatic treatment during the purification process. The amplification of cells and their growth in spinner bottles takes ~13 d; the tubulin purification takes 6-7 h. The tubulin can be used in total internal reflection fluorescence (TIRF)-microscopy-based experiments or pelleting assays for the investigation of intrinsic properties of microtubules and their interactions with associated proteins.


Assuntos
Processamento de Proteína Pós-Traducional/genética , Tubulina (Proteína)/química , Tubulina (Proteína)/isolamento & purificação , Animais , Reatores Biológicos , Química Encefálica , Linhagem Celular , Células HeLa , Humanos , Camundongos , Polimerização , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Ultracentrifugação
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