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1.
Phys Rev Lett ; 125(5): 058101, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32794890

RESUMO

Diffusion of tracer particles in the cytoplasm of mammalian cells is often anomalous with a marked heterogeneity even within individual particle trajectories. Despite considerable efforts, the mechanisms behind these observations have remained largely elusive. To tackle this problem, we performed extensive single-particle tracking experiments on quantum dots in the cytoplasm of living mammalian cells at varying conditions. Analyses of the trajectories reveal a strong, microtubule-dependent subdiffusion with antipersistent increments and a substantial heterogeneity. Furthermore, particles stochastically switch between different mobility states, most likely due to transient associations with the cytoskeleton-shaken endoplasmic reticulum network. Comparison to simulations highlight that all experimental observations can be fully described by an intermittent fractional Brownian motion, alternating between two states of different mobility.


Assuntos
Citoplasma/metabolismo , Modelos Biológicos , Citoesqueleto de Actina/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Simulação por Computador , Citocalasina D/farmacologia , Citoplasma/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Difusão , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nocodazol/farmacologia , Pontos Quânticos , Processos Estocásticos , Tiazolidinas/farmacologia
2.
Nat Commun ; 11(1): 3765, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32724196

RESUMO

Microtubules are dynamic tubulin polymers responsible for many cellular processes, including the capture and segregation of chromosomes during mitosis. In contrast to textbook models of tubulin self-assembly, we have recently demonstrated that microtubules elongate by addition of bent guanosine triphosphate tubulin to the tips of curving protofilaments. Here we explore this mechanism of microtubule growth using Brownian dynamics modeling and electron cryotomography. The previously described flaring shapes of growing microtubule tips are remarkably consistent under various assembly conditions, including different tubulin concentrations, the presence or absence of a polymerization catalyst or tubulin-binding drugs. Simulations indicate that development of substantial forces during microtubule growth and shortening requires a high activation energy barrier in lateral tubulin-tubulin interactions. Modeling offers a mechanism to explain kinetochore coupling to growing microtubule tips under assisting force, and it predicts a load-dependent acceleration of microtubule assembly, providing a role for the flared morphology of growing microtubule ends.


Assuntos
Microtúbulos/metabolismo , Modelos Biológicos , Tubulina (Proteína)/metabolismo , Animais , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Simulação de Dinâmica Molecular , Polimerização/efeitos dos fármacos , Suínos , Tubulina (Proteína)/isolamento & purificação , Tubulina (Proteína)/ultraestrutura , Moduladores de Tubulina/farmacologia
3.
Proc Natl Acad Sci U S A ; 117(28): 16154-16159, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601228

RESUMO

The metaphase spindle is a dynamic structure orchestrating chromosome segregation during cell division. Recently, soft matter approaches have shown that the spindle behaves as an active liquid crystal. Still, it remains unclear how active force generation contributes to its characteristic spindle-like shape. Here we combine theory and experiments to show that molecular motor-driven forces shape the structure through a barreling-type instability. We test our physical model by titrating dynein activity in Xenopus egg extract spindles and quantifying the shape and microtubule orientation. We conclude that spindles are shaped by the interplay between surface tension, nematic elasticity, and motor-driven active forces. Our study reveals how motor proteins can mold liquid crystalline droplets and has implications for the design of active soft materials.


Assuntos
Metáfase/fisiologia , Fuso Acromático/fisiologia , Animais , Fenômenos Biomecânicos , Dineínas/antagonistas & inibidores , Dineínas/metabolismo , Elasticidade , Cristais Líquidos , Metáfase/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Mitose , Fuso Acromático/química , Fuso Acromático/efeitos dos fármacos , Tensão Superficial , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/metabolismo , Xenopus laevis
4.
Mol Cell ; 79(1): 191-198.e3, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32619469

RESUMO

We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células , Glicina/análogos & derivados , Microtúbulos/efeitos dos fármacos , Neoplasias/patologia , Preparações Farmacêuticas/metabolismo , Sulfonas/farmacologia , Tubulina (Proteína)/metabolismo , Células Cultivadas , Cristalografia por Raios X , Contaminação de Medicamentos , Glicina/farmacologia , Humanos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Preparações Farmacêuticas/química , Conformação Proteica , Tubulina (Proteína)/química , Tubulina (Proteína)/genética
5.
Nucleic Acids Res ; 48(14): e83, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32526036

RESUMO

Mass spectrometry (MS)-based quantitative proteomics experiments frequently generate data with missing values, which may profoundly affect downstream analyses. A wide variety of imputation methods have been established to deal with the missing-value issue. To date, however, there is a scarcity of efficient, systematic, and easy-to-handle tools that are tailored for proteomics community. Herein, we developed a user-friendly and powerful stand-alone software, NAguideR, to enable implementation and evaluation of different missing value methods offered by 23 widely used missing-value imputation algorithms. NAguideR further evaluates data imputation results through classic computational criteria and, unprecedentedly, proteomic empirical criteria, such as quantitative consistency between different charge-states of the same peptide, different peptides belonging to the same proteins, and individual proteins participating protein complexes and functional interactions. We applied NAguideR into three label-free proteomic datasets featuring peptide-level, protein-level, and phosphoproteomic variables respectively, all generated by data independent acquisition mass spectrometry (DIA-MS) with substantial biological replicates. The results indicate that NAguideR is able to discriminate the optimal imputation methods that are facilitating DIA-MS experiments over those sub-optimal and low-performance algorithms. NAguideR further provides downloadable tables and figures supporting flexible data analysis and interpretation. NAguideR is freely available at http://www.omicsolution.org/wukong/NAguideR/ and the source code: https://github.com/wangshisheng/NAguideR/.


Assuntos
Proteômica/métodos , Software , Células/efeitos dos fármacos , Simulação por Computador , Conjuntos de Dados como Assunto , Formaldeído/farmacologia , Humanos , Espectrometria de Massas , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , Precursores de Proteínas/química
6.
Parasitol Res ; 119(8): 2695-2702, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32556538

RESUMO

Opisthorchis felineus is a trematode flatworm that parasitises mammals, including humans, and is mainly spread throughout Eastern Europe and Western Siberia. The main drug used in treatment of opisthorchiasis and other trematode and cestode infestations is praziquantel (PZQ). We provide a possible explanation of PZQ-mediated tegument disruption. The idea is that the nature of tegument disruption is related to failure of surface renovation due to insufficiency of microtubule transport of vesicles. This insufficiency arises from microtubule destabilisation, which in the medium term leads to the decrease in tubulins alpha, beta and dynein mRNA amounts and deficiency of the corresponding proteins. We also found the upregulation of cGMP-dependent protein kinase gene, and we concluded that its protein product helped to overcome the effect of praziquantel and might be a promising target for combined anthelmintic therapy with PZQ. We concluded that function of saposin-like protein 2 (SAP2) is unlikely associated with membrane fusion, and SAP2 is probably able to bind some type of hydrophobic compounds including praziquantel.


Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Helminto/genética , Opisthorchis/efeitos dos fármacos , Praziquantel/farmacologia , Animais , Antiplatelmínticos/farmacologia , Antiplatelmínticos/uso terapêutico , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/genética , Opistorquíase/tratamento farmacológico , Praziquantel/uso terapêutico
7.
Am J Physiol Lung Cell Mol Physiol ; 318(6): L1145-L1157, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32267731

RESUMO

We have demonstrated previously that intracellular transport is impaired in cystic fibrosis (CF) epithelial cells. This impairment is related to both growth and inflammatory regulation in CF cell and animal models. Understanding how transport in CF cells is regulated and identifying means to manipulate that regulation are key to identifying new therapies that can address key CF phenotypes. It was hypothesized that resveratrol could replicate these benefits since it interfaces with multiple pathways identified to affect microtubule regulation in CF. It was found that resveratrol treatment significantly restored intracellular transport as determined by monitoring both cholesterol distribution and the distribution of rab7-positive organelles in CF cells. This restoration of intracellular transport is due to correction of both microtubule formation rates and microtubule acetylation in cultured CF cell models and primary nasal epithelial cells. Mechanistically, the effect of resveratrol on microtubule regulation and intracellular transport was dependent on peroxisome proliferator-activated receptor-γ signaling and its ability to act as a pan-histone deacetylase (HDAC) inhibitor. Resveratrol represents a candidate compound with known anti-inflammatory properties that can restore both microtubule formation and acetylation in CF epithelial cells.


Assuntos
Fibrose Cística/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Espaço Intracelular/metabolismo , Resveratrol/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , Acetilação/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Carbazóis/farmacologia , Células Cultivadas , Colesterol/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Espaço Intracelular/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nariz/patologia , PPAR gama/antagonistas & inibidores , PPAR gama/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Resorcinóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sirtuínas/metabolismo , Estilbenos/farmacologia , Tubulina (Proteína)/metabolismo
8.
PLoS One ; 15(4): e0228771, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32255788

RESUMO

Hyperphosphorylated tau protein is a pathological hallmark of numerous neurodegenerative diseases and the level of tau pathology is correlated with the degree of cognitive impairment. Tau hyper-phosphorylation is thought to be an early initiating event in the cascade leading to tau toxicity and neuronal death. Inhibition of tau phosphorylation therefore represents an attractive therapeutic strategy. However, the widespread expression of most kinases and promiscuity of their substrates, along with poor selectivity of most kinase inhibitors, have resulted in systemic toxicities that have limited the advancement of tau kinase inhibitors into the clinic. We therefore focused on the CNS-specific tau kinase, TTBK1, and investigated whether selective inhibition of this kinase could represent a viable approach to targeting tau phosphorylation in disease. In the current study, we demonstrate that TTBK1 regulates tau phosphorylation using overexpression or knockdown of this kinase in heterologous cells and primary neurons. Importantly, we find that TTBK1-specific phosphorylation of tau leads to a loss of normal protein function including a decrease in tau-tubulin binding and deficits in tubulin polymerization. We then describe the use of a novel, selective small molecule antagonist, BIIB-TTBK1i, to study the acute effects of TTBK1 inhibition on tau phosphorylation in vivo. We demonstrate substantial lowering of tau phosphorylation at multiple sites implicated in disease, suggesting that TTBK1 inhibitors may represent an exciting new approach in the search for neurodegenerative disease therapies.


Assuntos
Doenças do Sistema Nervoso Central/enzimologia , Doenças do Sistema Nervoso Central/patologia , Sistema Nervoso Central/enzimologia , Sistema Nervoso Central/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas tau/metabolismo , Animais , Células Cultivadas , Masculino , Camundongos Endogâmicos C57BL , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Especificidade de Órgãos , Fosforilação/efeitos dos fármacos , Polimerização , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Tubulina (Proteína)/metabolismo
9.
Toxicology ; 439: 152466, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32315717

RESUMO

Glyphosate is the most popular herbicide used in modern agriculture, and its use has been increasing substantially since its introduction. Accordingly, glyphosate exposure from food and water, the environment, and accidental and occupational venues has also increased. Recent studies have demonstrated a relationship between glyphosate exposure and a number of disorders such as cancer, immune and metabolic disorders, endocrine disruption, imbalance of intestinal flora, cardiovascular disease, and infertility; these results have given glyphosate a considerable amount of media and scientific attention. Notably, glyphosate is a powerful metal chelator, which could help explain some of its effects. Recently, our findings on 2,3-dimercapto-1-propanesulfonic acid, another metal chelator, showed deterioration of oocyte quality. Here, to generalize, we investigated the effects of glyphosate (0 - 300 µM) on metaphase II mouse oocyte quality and embryo damage to obtain insight on its mechanisms of cellular action and the tolerance of oocytes and embryos towards this chemical. Our work shows for the first time that glyphosate exposure impairs metaphase II mouse oocyte quality via two mechanisms: 1) disruption of the microtubule organizing center and chromosomes such as anomalous pericentrin formation, spindle fiber destruction and disappearance, and defective chromosomal alignment and 2) substantial depletion of intracellular zinc bioavailability and enhancement of reactive oxygen species accumulation. Similar effects were found in embryos. These results may help clarify the effects of glyphosate exposure on female fertility and provide counseling and preventative steps for excessive glyphosate intake and resulting oxidative stress and reduced zinc bioavailability.


Assuntos
Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Glicina/análogos & derivados , Herbicidas/toxicidade , Metáfase/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Zinco/metabolismo , Animais , Cromossomos/efeitos dos fármacos , Feminino , Glicina/toxicidade , Infertilidade Feminina/induzido quimicamente , Infertilidade Feminina/patologia , Camundongos , Microtúbulos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Gravidez , Fuso Acromático/efeitos dos fármacos
10.
Mol Pharmacol ; 97(6): 409-422, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32241960

RESUMO

The microtubule-binding taxanes, docetaxel and cabazitaxel, are administered intravenously for the treatment of castration-resistant prostate cancer (CRPC) as the oral administration of these drugs is largely hampered by their low and highly variable bioavailabilities. Using a simple, rapid, and environmentally friendly microwave-assisted protocol, we have synthesized a number of 3,5-bis(styryl)pyrazoles 2a-l, thus allowing for their screening for antiproliferative activity in the androgen-independent PC3 prostate cancer cell line. Surprisingly, two of these structurally simple 3,5-bis(styryl)pyrazoles (2a and 2l) had concentrations which gave 50% of the maximal inhibition of cell proliferation (GI50) in the low micromolar range in the PC3 cell line and were thus selected for extensive further biologic evaluation (apoptosis and cell cycle analysis, and effects on tubulin and microtubules). Our findings from these studies show that 3,5-bis[(1E)-2(2,6-dichlorophenyl)ethenyl]-1H-pyrazole 2l 1) caused significant effects on the cell cycle in PC3 cells, with the vast majority of treated cells in the G2/M phase (89%); 2) induces cell death in PC3 cells even after the removal of the compound; 3) binds to tubulin [dissociation constant (Kd) 0.4 ± 0.1 µM] and inhibits tubulin polymerization in vitro; 4) had no effect upon the polymerization of the bacterial cell division protein FtsZ (a homolog of tubulin); 5) is competitive with paclitaxel for binding to tubulin but not with vinblastine, crocin, or colchicine; and 6) leads to microtubule depolymerization in PC3 cells. Taken together, these results suggest that 3,5-bis(styryl)pyrazoles warrant further investigation as lead compounds for the treatment of CRPC. SIGNIFICANCE STATEMENT: The taxanes are important components of prostate cancer chemotherapy regimens, but their oral administration is hampered by very low and highly variable oral bioavailabilities resulting from their poor absorption, poor solubility, high first-pass metabolism, and efficient efflux by P-glycoprotein. New chemical entities for the treatment of prostate cancer are thus required, and we report here the synthesis and investigation of the mechanism of action of some bis(styryl)pyrazoles, demonstrating their potential as lead compounds for the treatment of prostate cancer.


Assuntos
Antineoplásicos/uso terapêutico , Chumbo/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Pirazóis/uso terapêutico , Tubulina (Proteína)/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Sítios de Ligação , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Estabilidade de Medicamentos , Humanos , Chumbo/química , Masculino , Microtúbulos/efeitos dos fármacos , Modelos Moleculares , Células PC-3 , Pirazóis/síntese química , Pirazóis/química
11.
Cell Prolif ; 53(4): e12749, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32167212

RESUMO

OBJECTIVES: Given that autophagy inhibition is a feasible way to enhance sensitivity of cancer cells towards chemotherapeutic agents, identifying potent autophagy inhibitor has obvious clinical relevance. Here, we investigated ability of TN-16, a microtubule disrupting agent, on modulation of autophagic flux and its significance in promoting in vitro and in vivo cancer cell death. MATERIALS AND METHODS: The effect of TN-16 on cancer cell proliferation, cell division, autophagic process and apoptotic signalling was assessed by various biochemical (Western blot and SRB assay), morphological (TEM, SEM, confocal microscopy) and flowcytometric assays. In vivo anti-tumour efficacy of TN-16 was investigated in syngeneic mouse model of breast cancer. RESULTS: TN-16 inhibited cancer cell proliferation by impairing late-stage autophagy and induction of apoptosis. Inhibition of autophagic flux was demonstrated by accumulation of autophagy-specific substrate p62 and lack of additional LC3-II turnover in the presence of lysosomotropic agent. The effect was validated by confocal micrographs showing diminished autophagosome-lysosome fusion. Further studies revealed that TN-16-mediated inhibition of autophagic flux promotes apoptotic cell death. Consistent with in vitro data, results of our in vivo study revealed that TN-16-mediated tumour growth suppression is associated with blockade of autophagic flux and enhanced apoptosis. CONCLUSIONS: Our data signify that TN-16 is a potent autophagy flux inhibitor and might be suitable for (pre-) clinical use as standard inhibitor of autophagy with anti-cancer activity.


Assuntos
Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Pirrolidinonas/uso terapêutico , Moduladores de Tubulina/uso terapêutico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Pirrolidinonas/farmacologia , Moduladores de Tubulina/farmacologia
12.
Chem Biol Interact ; 323: 109074, 2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-32217108

RESUMO

Non-small-cell lung cancer (NSCLC) is one of the common malignant tumors, and multidrug resistance (MDR) and tumor metastasis limit the anticancer effect of NSCLC. Therefore, it is necessary to develop new anticancer drug that can inhibit MDR and metastasis of NSCLC. In the present study, we found that 5-(2-chlorophenyl)-4-(4-(3,5-dimethoxyphenyl)piperazine-1-carbonyl)-2H-1,2,3- triazole (MAY) displayed strong cytotoxic effect on A549 and taxol-resistant A549 cells (A549/Taxol cells). We further discovered that MAY led to G2/M phase arrest by inhibiting microtubule polymerization in both cells. Then MAY caused apoptosis by the mitochondrial pathway in A549 cells and through the extrinsic pathway in A549/Taxol cells. Interestingly, MAY was not a substrate for P-glycoprotein (P-gp), which was highly expressed in A549/Taxol cells, and MAY inhibited the expression and efflux function of P-gp. Furthermore, MAY inhibited epithelial-mesenchymal transition (EMT) by targeting Twist1 in A549/Taxol cells. In summary, our results suggest that MAY induces apoptosis in A549 and A549/Taxol cells and inhibits EMT in A549/Taxol cells. These findings suggest that MAY could provide a promising method for the treatment of NSCLC, especially for the treatment of resistant NSCLC.


Assuntos
Apoptose/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Paclitaxel/farmacologia , Triazóis/farmacologia , Moduladores de Tubulina/farmacologia , Células A549 , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Paclitaxel/química , Polimerização , Transdução de Sinais/efeitos dos fármacos , Triazóis/química , Moduladores de Tubulina/química , Proteína 1 Relacionada a Twist/metabolismo
13.
Int Heart J ; 61(2): 355-363, 2020 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-32173700

RESUMO

Heart failure (HF) is a disease with high morbidity and mortality. In patients with HF, decreased cardiac output and blood redistribution results in decreased intestinal perfusion and destruction of intestinal barrier. Microorganisms and endotoxins can migrate into the blood circulation, aggravating systemic inflammation and HF. Trimethylamine N-oxide (TMAO) is highly closed to the occurrence of HF. However, the exact mechanism between TMAO and HF remains unclear.To investigate the role of TMAO in transverse-tubule (T-tubule) in the cultured cardiomyocytes.T-tubule imaging and analysis detected T-tubule network in cardiomyocytes. Ca2+ handling dysfunction was identified by confocal Ca2+ imaging. Tubulin densification and polymerization were assessed by western blot and immunofluorescent staining of cardiomyocytes.TMAO induced T-tubule network damage in cardiomyocytes and Ca2+ handling dysfunction in cardiomyocytes under the TMAO stress via promoting tubulin densification and polymerization and therefore Junctophilin-2 (JPH2) redistribution. Mice treated with TMAO represented cardiac dysfunction and T-tubule network disorganization.TMAO impairs cardiac function via the promotion of tubulin polymerization, subsequent translocation of JPH2, and T-tubule remodeling, which provides a novel mechanism for the relationship between HF and elevated TMAO.


Assuntos
Insuficiência Cardíaca/metabolismo , Metilaminas/toxicidade , Microtúbulos/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Acoplamento Excitação-Contração , Insuficiência Cardíaca/etiologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Microtúbulos/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Tubulina (Proteína)/metabolismo
14.
Chemosphere ; 248: 126066, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32050317

RESUMO

Silver nanoparticles (AgNPs) are an emerging contaminant, currently considered to be a significant potential risk to the coastal environment. To further test potential risk, and to determine effect concentrations and sensitive response parameters, toxic effects of environmentally relevant AgNP concentrations on the seagrass Cymodocea nodosa were evaluated. Alterations of the cytoskeleton, endoplasmic reticulum, ultrastructure, photosystem II function, oxidative stress markers, cell viability, and leaf, rhizome and root elongation in C. nodosa exposed to AgNP concentrations (0.0002-0.2 mg L-1) under laboratory conditions for 8 days were examined. An increase in H2O2 level, indicating oxidative stress, occurred after the 4th day even at 0.0002 mg L-1. Increased antioxidant enzyme activity, potentially contributing to H2O2 level decline at the end of the experiment, and reduced protein content were also observed. Actin filaments started to diminish on the 6th day at 0.02 mg L-1; microtubule, endoplasmic reticulum, chloroplast and mitochondrion disturbance appeared after 8 days at 0.02 mg L-1, while toxic effects were generally more acute at 0.2 mg L-1. A dose-dependent leaf elongation inhibition was also observed; as for juvenile leaves, toxicity index increased from 2.8 to 40.7% with concentration. Hydrogen peroxide (H2O2) overproduction and actin filament disruption appeared to be the most sensitive response parameters, and thus could be utilized as early warning indicators of risk to seagrass meadows. A risk quotient of 1.33 was calculated, confirming previous findings, that AgNPs may pose a significant risk to the coastal environment.


Assuntos
Alismatales/fisiologia , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Poluentes Químicos da Água/toxicidade , Alismatales/efeitos dos fármacos , Alismatales/ultraestrutura , Antioxidantes/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Microtúbulos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/metabolismo
15.
Chem Biol Interact ; 315: 108906, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31760042

RESUMO

The microtubule inhibitor (MTI) class of chemotherapeutics provide an effective treatment for several different types of cancers, however, severe chemotherapy-induced peripheral neuropathy (CIPN) is a major dose limiting toxicity in patients that limits their use. While CIPN was predicted with MTIs based on histopathology and functional effects in non-clinical toxicology studies, these investigations often require large numbers of animals and long term studies. As in vitro MT assays have been used for decades to study mechanisms of efficacy, we hypothesized that those same assays could be used to study mechanisms of peripheral neuropathy and predict severe CIPN. We analyzed published data on in vitro microtubule (MT) properties for different MTIs that cause varying levels of peripheral neuropathy in patients. Eribulin, vinorelbine and vinfluinine, which all have less severe CIPN than the vinca alkaloids or taxanes, have unique MT properties consisting of reduced affinity and limited binding to MTs (i.e. bind only to the ends and not along the length). Binding more potently to tubulin in the absence of neuronal BIII tubulin was also observed with eribulin and may suggest specificity for tumor tubulin over neuronal tubulin. These are possible mechanisms for causing less severe deleterious effects on MTs in peripheral nerves leading to reduced severity of CIPN. Our analyses demonstrated that in vitro tools used to study the mechanisms of action in inducing severe CIPN (i.e MTI interactions with MTs) warrant further investigation and may be useful for developing next generation MTIs with reduced CIPN.


Assuntos
Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Microtúbulos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Moduladores de Tubulina/efeitos adversos , Moduladores de Tubulina/uso terapêutico , Animais , Humanos , Microtúbulos/metabolismo , Neoplasias/metabolismo , Doenças do Sistema Nervoso Periférico/metabolismo , Tubulina (Proteína)/metabolismo
16.
Eur J Med Chem ; 186: 111865, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31735573

RESUMO

We here report the discovery of isoquinoline-based biaryls as a new scaffold for colchicine domain tubulin inhibitors. Colchicinoid inhibitors offer highly desirable cytotoxic and vascular disrupting bioactivities, but their further development requires improving in vivo robustness and tolerability: properties that both depend on the scaffold structure employed. We have developed isoquinoline-based biaryls as a novel scaffold for high-potency tubulin inhibitors, with excellent robustness, druglikeness, and facile late-stage structural diversification, accessible through a tolerant synthetic route. We confirmed their bioactivity mechanism in vitro, developed soluble prodrugs, and established safe in vivo dosing in mice. By addressing several problems facing the current families of inhibitors, we expect that this new scaffold will find a range of in vivo applications towards translational use in cancer therapy.


Assuntos
Antineoplásicos/farmacologia , Isoquinolinas/farmacologia , Microtúbulos/efeitos dos fármacos , Moduladores de Tubulina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HL-60 , Células HeLa , Humanos , Isoquinolinas/síntese química , Isoquinolinas/química , Microscopia Confocal , Microtúbulos/metabolismo , Estrutura Molecular , Polimerização/efeitos dos fármacos , Relação Estrutura-Atividade , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/química
17.
Eur J Med Chem ; 187: 111949, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31830637

RESUMO

A combination therapeutic regimen via introducing tryptophan 2,3-dioxygenase inhibitors into microtubule inhibitors was performed and evaluated for their antitumor activity. Thereinto, HT2, composed of combretastatin A-4 (CA-4) and tryptophan-2,3-dioxygenase (TDO) inhibitor by a linker, displayed the most potent activity with 10-fold higher than its parent CA-4 against HepG2, A549 and HCT-116 cancer cell lines. Mechanism studies suggested that HT2 inhibited tubulin polymerization and cell migration, caused G2 phase arrest, induced apoptosis by mitochondrial mediated apoptotic pathway, concurrent depolarized the mitochondria membrane potentials and caused reactive oxygen species (ROS) production in HepG2 cells. Moreover, HT2 could enhance T-cell immune responses in vitro by releasing a TDO inhibitor to suppress TDO expression and blockade kynurenine production. As expected, HT2 could remarkably promote the antitumor activity of CA-4 in either immunocompetent H22 or immunodeficient A549 tumor xenograft models without observable toxic effects. More importantly, HT2 increased the level of splenic and tumor-infiltrated T cells and in turn effectively boosted the inhibition effect in H22 xenografted tumor growth. Collectively, this immunochemotherapeutic strategy can be applied to promote chemotherapeutic effect.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Fatores Imunológicos/farmacologia , Microtúbulos/efeitos dos fármacos , Triptofano Oxigenase/antagonistas & inibidores , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Fatores Imunológicos/síntese química , Fatores Imunológicos/química , Imunomodulação/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Polimerização/efeitos dos fármacos , Relação Estrutura-Atividade , Triptofano Oxigenase/metabolismo
18.
Oncogene ; 39(2): 454-468, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31492900

RESUMO

The nuclear transport receptor importin-ß/karyopherin-ß1 is overexpressed in cancers that display genomic instability. It is regarded as a promising cancer target and inhibitors are being developed. In addition to its role in nucleo-cytoplasmic transport, importin-ß regulates mitosis, but the programmes and pathways in which it operates are defined only in part. To unravel importin-ß's mitotic functions we have developed cell lines expressing either wild-type or a mutant importin-ß form in characterised residues required for nucleoporin binding. Both forms similarly disrupted spindle pole organisation, while only wild-type importin-ß affected microtubule plus-end function and microtubule stability. A proteome-wide search for differential interactors identified a set of spindle regulators sensitive to mutations in the nucleoporin-binding region. Among those, HURP (hepatoma up-regulated protein) is an importin-ß interactor and a microtubule-stabilising factor. We found that induction of wild type, but not mutant importin-ß, under the same conditions that destabilise mitotic microtubules, delocalised HURP, indicating that the spatial distribution of HURP along the spindle requires importin-ß's nucleoporin-binding residues. Concomitantly, importin-ß overexpression sensitises cells to taxanes and synergistically increases mitotic cell death. Thus, the nucleoporin-binding domain is dispensable for importin-ß function in spindle pole organisation, but regulates microtubule stability, at least in part via HURP, and renders cells vulnerable to certain microtubule-targeting drugs.


Assuntos
Hidrocarbonetos Aromáticos com Pontes/farmacologia , Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Taxoides/farmacologia , beta Carioferinas/química , beta Carioferinas/metabolismo , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Microtúbulos/efeitos dos fármacos , Paclitaxel/farmacologia , Ligação Proteica
19.
Ecotoxicol Environ Saf ; 189: 109925, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31855841

RESUMO

Information on silver nanoparticle (AgNP) phytotoxicity on seagrasses is provided for the first time. Toxic effects of environmentally relevant AgNP concentrations on Halophila stipulacea were assessed to identify sensitive biomarkers, to determine threshold effect concentrations and to evaluate potential risks. Potential alterations in the cytoskeleton, endoplasmic reticulum, cell ultrastructure and viability, oxidative stress parameters and elongation in H. stipulacea leaves exposed to AgNP concentrations ranging from 0.0002 to 0.2 mg L-1 for 8 days were examined. The first signs of actin filament (AF) response in differentiating cells, exhibiting disorientation and slight bundling, were observed on the 4th day at 0.0002 mg L-1, while at the end of the experiment and at the higher concentrations, AFs were extremely bundled. Endoplasmic reticulum was affected in meristematic and differentiating cells; massive aggregations and loss of the "grainy" structure were observed, initially on the 6th day at 0.002 mg L-1. Effects on microtubules were detected on the last day at 0.2 mg L-1. An increase in H2O2 levels on the 4th and/or 6th day even at 0.0002 mg L-1 was followed by a decrease on, or up to the last day. On the 6th day at the lowest concentration, elevated malondialdehyde content, and superoxide dismutase and peroxidase activity were detected, indicating oxidative damage and antioxidant defense mechanism activation. Dead epidermal cells mainly occurred at 0.02 and 0.2 mg L-1, while no dead vein cells were detected. A significant inhibition in leaf elongation was observed only at 0.2 mg L-1. Therefore, AF disturbance in differentiating leaf cells, being a susceptible response parameter, could be regarded as an early warning indicator of risk posed by AgNPs to H. stipulacea meadows, while most of the remaining parameters examined also constitute useful biomarkers. The lowest observed effect concentration (0.0002 mg L-1), being within the range of environmentally relevant AgNPs concentrations, suggests the possibility of negative impacts of AgNPs on seagrass health. A risk quotient of 1.33 was calculated, indicating that AgNPs may pose a significant potential risk to the coastal environment. The data presented highlight the importance of future research to further investigate the seagrass-AgNP interactions, stress the need for a refinement of the environmental risk assessment of AgNPs and could be utilized for the design of biomonitoring programs for rational management of the coastal environment.


Assuntos
Hydrocharitaceae/fisiologia , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Antioxidantes/farmacologia , Citoesqueleto/efeitos dos fármacos , Hydrocharitaceae/efeitos dos fármacos , Peróxido de Hidrogênio , Malondialdeído/farmacologia , Microtúbulos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Superóxido Dismutase/análise
20.
Biochim Biophys Acta Mol Basis Dis ; 1866(1): 165581, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31672549

RESUMO

Exposure to environmental toxins, including hydrocarbon solvents, increases the risk of developing Parkinson's disease. An emergent hypothesis considers microtubule dysfunction as one of the crucial events in triggering neuronal degeneration in Parkinson's disease. Here, we used 2,5-hexanedione (2,5-HD), the toxic metabolite of n-hexane, to analyse the early effects of toxin-induced neurodegeneration on the cytoskeleton in multiple model systems. In PC12 cells differentiated with nerve growth factor for 5 days, we found that 2,5-HD treatment affected all the cytoskeletal components. Moreover, we observed alterations in microtubule distribution and stability, in addition to the imbalance of post-translational modifications of α-tubulin. Similar defects were also found in vivo in 2,5-HD-intoxicated mice. Interestingly, we also found that 2,5-HD exposure induced significant changes in microtubule stability in human skin fibroblasts obtained from Parkinson's disease patients harbouring mutations in PRKN gene, whereas it was ineffective in healthy donor fibroblasts, suggesting that the genetic background may really make the difference in microtubule susceptibility to this environmental Parkinson's disease-related toxin. In conclusion, by showing the imbalance between dynamic and stable microtubules in hydrocarbon-induced parkinsonism, our data support the crucial role of microtubule defects in triggering neurodegeneration.


Assuntos
Hexanonas/farmacologia , Microtúbulos/efeitos dos fármacos , Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Animais , Linhagem Celular , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Camundongos , Microtúbulos/metabolismo , Fatores de Crescimento Neural/metabolismo , Células PC12 , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/metabolismo , Ratos , Tubulina (Proteína)/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
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